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.

A Black Swan in clinical laboratory practice: the analytical error due to interferences in immunoassay methods.

It is well known that the results of immunoassay methods can be affected by specific or non-specific interferences, ranging from 0.4% to 4.0%. The presence of interference may greatly compromise the accuracy of immunoassay analyses causing an error in the measurement, producing false-positive or false-negative results. From a clinical point of view, these analytical errors may have serious implications for patient care because they can cause misdiagnosis or inappropriate treatment. Unfortunately, it is a very difficult task to identify the irregular analytical errors related to immunoassay methods because they are not detectable by normal laboratory quality control procedures, are reproducible within the test system, may be clinically plausible and are relatively rare. The first line of defense against erroneous results is to use in laboratory practice only immunoassay systems with the highest level of robustness against interference. The second line of defense is always taking into account the possibility of interference in immunoassay results. A correct approach should be addressed on identification of samples at high risk of interference. The attainment of this goal requires a critical review of the test result in relation to patient's clinical conditions and literature data, taking into account the analytical characteristics of the immunoassay system. The experts in immunoassay systems should make every effort to find some specific and reliable quality indicators for irregular analytical errors in order to better detect and monitor erroneous immunoassay results due to specific or non-specific interferences.

Pilot study on harmonization of cardiac troponin I immunoassays using patients and quality control plasma samples. On behalf of the Italian Section of the European Ligand Assay Society (ELAS) and of the Study Group on Cardiovascular Biomarkers of the Società Italiana di Biochimica Clinica (SIBioC).

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.

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.

State of the art of aldosterone immunoassays. A multicenter collaborative study on the behalf of the Cardiovascular Biomarkers Study Group of the Italian Section of European Society of Ligand Assay (ELAS) and Società Italiana di Biochimica Clinica (SIBIOC).

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.

Harmonization protocols for thyroid stimulating hormone (TSH) immunoassays: different approaches based on the consensus mean value.

The lack of interchangeable laboratory results and consensus in current practices has underpinned greater attention to standardization and harmonization projects. In the area of method standardization and harmonization, there is considerable debate about how best to achieve comparability of measurement for immunoassays, and in particular heterogeneous proteins. The term standardization should be used only when comparable results among measurement procedures are based on calibration traceability to the International System of Units (SI unit) using a reference measurement procedure (RMP). Recently, it has been promoted the harmonization of methods for many immunoassays, and in particular for thyreotropin (TSH), as accepted RMPs are not available. In a recent paper published in this journal, a group of well-recognized authors used a complex statistical approach in order to reduce variability between the results observed with the 14 TSH immunoassay methods tested in their study. Here we provide data demonstrating that data from an external quality assessment (EQA) study allow similar results to those obtained using the reported statistical approach.

State of the art of BNP and NT-proBNP immunoassays: the CardioOrmoCheck study.

To evaluate differences in analytical performance and clinical results of BNP and NT-proBNP immunoassays, a proficiency testing program, called CardioOrmoCheck study, has been organized since 2005 under the patronage of the Study Group of the Cardiovascular Biomarkers of the Italian Society of Clinical Biochemistry (SIBIOC). On average more than 100 Italian laboratories were involved in the annual 2005-2011 cycles. In total, 72 study samples were distributed and measured by participant laboratories for a total of 6706 results. A great difference in between-method variability was found between BNP (43.0 CV%) and NT-proBNP (8.7 CV%) immunoassays. However, with the only exception of the POCT method for BNP assay, all immunoassay methods showed an imprecision ≤ 10 CV% at the cut-off levels (i.e. 100 ng/L for BNP and 400 ng/L for NT-proBNP assay, respectively). Furthermore, CardioOrmoCheck study demonstrated that the most popular BNP immunoassays are affected by large systematic differences (on average more than 2 folds between TRIAGE Beckman-Coulter and ADVIA Centaur Siemens methods), while the agreement between NT-proBNP methods was better. CardioOrmoCheck study demonstrates that there are marked differences in analytical performance and measured values in particular among commercial methods for BNP assay. These findings suggest that it may be not reasonable to recommend identical cut-off or decision values for all BNP immunoassays.

Multicentre comparison of free thyroid hormones immunoassays: the Immunocheck study.

BACKGROUND: In order to evaluate whether there is still present a very large variability in measured values and analytical performance among different free thyroid (FT) hormone immunoassays, we considered the results derived from an External Quality Assessment (EQA) scheme, named Immunocheck. METHODS: The EQA Immonocheck study enrolled about 1000 participant laboratories, which measured the 54 quality control samples distributed in the three annual cycles (2007, 2008 and 2009). Participant laboratories produced a total of 30,476 results for FT3 and 31,351 for FT4, respectively. RESULTS: The results recovered during the EQA cycles allowed the estimation of assay imprecision (i.e., within-method and between-laboratories variability). On average, control samples with lower free triiodothyronine (FT3) and free thyroxine (FT4) concentrations showed higher imprecision values (CV%) than those with levels within or above the normal range. The agreement among the results produced by different methods was estimated by computing the percent differences (i.e., percent bias values) between the concentrations measured by the methods and the mean of all collected results (i.e., consensus mean). CONCLUSIONS: The results of our study demonstrate that a large variability in measured values is still present among different free thyroid hormone immunoassays. Indeed, some immunoassays for both FT3 and FT4 measurement showed percent bias values compared to the consensus mean >20%. Laboratories should inform the clinicians about analytical performance and reference limits of the method used. Furthermore, the clinicians should avoid the use of different methods in the follow-up of patients.

Proficiency testing project for brain natriuretic peptide (BNP) and the N-terminal part of the propeptide of BNP (NT-proBNP) immunoassays: the CardioOrmocheck study.

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.

Comparison between analytical performances of polyclonal and monoclonal electrochemiluminescence immunoassays for NT-proBNP.

BACKGROUND: We evaluated the analytical characteristics of electrochemiluminescence (ECLIA) immunoassay for NT-proBNP (proBNP II, Roche Diagnostics, Germany) and compared its analytical performance to that of the previous polyclonal method. METHODS: We measured NT-proBNP in EDTA plasma samples of 177 consecutive cardiac patients (69 females and 108 males; mean age 62.2+/-16.4 years, range 13 to 96 years) with monoclonal and polyclonal ECLIA methods following manufacturer's instructions using an Elecsys 2010 analyzer. RESULTS: Monoclonal ECLIA method for NT-proBNP assay showed an imprecision (CV%) lower than 3% at the cut-off value (i.e., 150 ng/L). No significant interference was found in plasma samples containing high levels of hemoglobin, triglycerides or bilirubin. EDTA plasma showed slightly, but significantly lower NT-proBNP values than serum (on average -6.3%) and lithium-heparinized plasma (on average -3.9%) samples. Finally, a very close linear regression was found between the NT-proBNP values found by either monoclonal or polyclonal ECLIA method (monoclonal ECLIA=-72.17+1.04 polyclonal ECLIA, n=177, R=0.993). CONCLUSIONS: The monoclonal ECLIA method showed very similar analytical characteristics with slightly lower NT-proBNP results (on average -2.5%) than the polyclonal ECLIA method. The difference between monoclonal and polyclonal methods seems to be too slight to change the reference range and decisional values for NT-proBNP assay, as previously assessed by the polyclonal ECLIA method.

Distribution of plasma cardiac troponin I values in healthy subjects: pathophysiological considerations.

BACKGROUND: The aim of this study was to evaluate the distribution of cardiac troponin I (cTnI) values, measured by the ADVIA TnI-Ultra method (Siemens Medical Solutions Diagnostics SrL) in healthy subjects and to characterize its relation to gender, age, as well as to N-terminal fragment of pro-brain natriuretic peptide (NT-proBNP). METHODS: A Caucasian population of 692 healthy subjects (311 males and 381 females) with a mean (SD) age of 45.3 (17.3) years [range 11-89 years; females 46.5 (17.3) years, males 43.8 (17.1) years] was enrolled. The presence of cardiac or systemic acute or chronic diseases was excluded by history and accurate clinical evaluation. RESULTS: A significant difference was found between the cTnI values in men and women (men: median 0.012 microg/L, range from undetectable values to 0.196 microg/L; women: median 0.008 microg/L, range from undetectable values to 0.130 microg/L; p<0.0001 by Mann-Whitney U-test). When a multiple regression analysis was performed, NT-proBNP, gender and age significantly contributed to the regression with cTnI (R=0.444, p<0.0001). CONCLUSIONS: Our data indicate that cut-off values, based on the 99th percentile of cTnI distribution in apparently healthy subjects, can significantly vary according to age and gender of the reference population.

Gender-related differences in oxidative stress levels among elderly patients with coronary artery disease.

OBJECTIVE: To evaluate whether gender-related differences exist concerning oxidative stress levels in aged patients with coronary artery disease (CAD). DESIGN: Case-control. SETTING: Clinical and research center. PATIENT(S): Elderly subjects of both genders with or without CAD. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Serum hydroperoxides (HP) as index of oxidative stress levels. RESULT(S): The HP levels were comparable in aged control subjects of both genders (376 +/- 20 arbitrary units [AU] in women, 333 +/- 19 AU in men) but significantly increased in CAD (456 +/- 15 AU) compared with all control subjects (357 +/- 14 AU). Moreover, among CAD patients, the HP levels were higher in women than in men (536 +/- 33 AU and 428 +/- 15 AU, respectively). Multivariate analysis, in which CAD represented the dependent variable, indicated that dyslipidemia was independently associated with CAD in men (odds ratio [OR] 5.8), whereas HP >50th percentile represented the only strong independent risk factor for CAD in elderly women (OR 8.4). CONCLUSION(S): Differences in oxidative stress levels between elderly males and females might provide a biochemical basis for the epidemiologic differences in CAD, which might help to open new opportunities in the management of patients with cardiovascular disease from a gender point of view.

Automation and validation of a fast method for the assessment of in vivo oxidative stress levels.

BACKGROUND: The d-ROMs test for the evaluation of serum hydroperoxides (HP) is simple, reliable, and cheap. Furthermore, it can easily be adapted to automated analyzers. Changing from the manual to an automated procedure allows the simultaneous processing of a large number of samples in a greatly reduced time, avoiding manual handling of samples and reagents and reducing variability sources. METHODS: This study was performed to adjust the manual procedure to a routine automated method in the clinical laboratory. We carried out the d-ROMs test in sera from 90 subjects of both sexes (34 men and 56 women) with age ranging from 20 to 80 years (mean 51+/-14 years). All subjects were free from acute or chronic inflammatory disease, immunological disease and history or evidence of malignancy. Subjects were not on vitamin and/or antioxidant therapies. RESULTS: The detection limit of the assay was 40 AU. Linearity was observed up to 475 AU. The recovery ranged between 97% and 105%. Within- and between-run imprecision was <5%. The mean HP value was 304+/-8 AU, with no significant difference between men (291+/-10 AU) and women (311+/-11 AU). A significant positive correlation was observed between age and HP in the whole population (r=0.4, p=0.0002). CONCLUSIONS: The automated test for the estimation of serum hydroperoxides represents a reliable and feasible procedure for increasing efficiency and reducing costs compared to the manual method, and is particularly suitable for evaluating oxidative stress in a variety of clinical conditions.

Evaluation of the analytical performance of the advanced method for cardiac troponin I for the AxSYM platform: comparison with the old method and the Access system.

BACKGROUND: The determination of cardiac troponins is routinely used for rule in/out, risk stratification, and follow-up of patients with acute coronary artery syndrome. We evaluated the analytical and clinical performance of the advanced immunoassay for troponin I (cTnI) carried out on an AxSYM platform (Abbott Diagnostic Division) and compared these characteristics to those of the previous version of this assay and to cTnI on the Access 2 immunoassay system (Beckman Coulter, Inc.). METHODS: We assayed plasma samples from healthy subjects (n=66) and cardiac patients (n=132) using AxSYM Plus system assays called the old (OLD AxSYM) and advanced TnI (ADV AxSYM) methods and using an Access system. RESULTS: An improvement in analytical sensitivity (detection limit) was observed for the advanced cTnI AxSYM compared to the previous method (0.014 vs. 0.31 microg/L), while the cTnI value for the 10% CV (i.e., functional sensitivity) was 0.41 microg/L for the ADV and 1.9 microg/L for the OLD method. The kinetics of cTnI release was similar, as evaluated in 25 patients with typical acute myocardial infarction (AMI). A close linear relationship was found between the two methods on the AxSYM system (OLD cTnI=7.436+6.858 ADV cTnI; R=0.968, n=214) and with the Access system (OLD AxSYM=7.154+7.9 Access, R=0.876, n=158; ADV AxSYM=0.23+1.209 Access, R=0.927, n=160). However, wide bias was found between the OLD and ADV AxSYM methods (mean difference 118.4 microg/L, p<0.0001), while more similar results were found between the ADV AxSYM and Access methods (mean difference 2.6 microg/L, corresponding to a mean percentage difference of 17%, p<0.0001). In 106 patients with symptomatic rheumatoid arthritis with high rheumatoid factor (RF) concentration, the mean cTnI measured by the ADV AxSYM method was 0.009+/-0.031 mug/L (range 0-0.23 microg/L) with a significant correlation (R=0.316, p=0.001) between cTnI and RF values. Furthermore, in 60 of these serum samples the cTnI concentration was also measured using the Access method; significant correlation with the values found by the ADV AxSYM method was observed (R=0.468, p=0.0002). CONCLUSIONS: The present study indicates that the AxSYM Troponin-I ADV immunoassay shows improved analytical sensitivity compared to the OLD AxSYM method, as well as very similar clinical results to those determined using the Access method.

Estrogen therapy effects on different vasoactive factors in recent postmenopausal healthy women.

BACKGROUND: To evaluate whether the route of estrogen therapy (ET) may affect the levels of different vasoactive factors in healthy recent post-menopausal women. METHODS: We conducted a cross-over study in 20 healthy nonsmoking women in recent postmenopause (1.8+/-0.1 years). Women received either 1-month oral-ET (O-ET, 2 mg oral micronized 17beta estradiol daily) or transdermal-ET regimen (T-ET, 17beta estradiol 1.5 mg gel daily) with a 1-month wash-out interval. Blood pressure, plasma levels of endothelin-1 (ET), 6-ketoPGF1a (6-ketoPG, prostacyclin metabolite), nitrite/nitrate (NOx), epinephrine (E) and norepinephrine (NE) and lipid profile were measured at baseline and after each treatment. RESULTS: Both regimens significantly reduced E (p<0.01) and NE levels (p<0.05). O-ET reduced low-density lipoproteins (LDL) levels (p<0.05) and increased NOx values (p<0.01). Neither regimen caused significant changes of ET or 6-ketoPG. CONCLUSIONS: Our results, obtained in healthy women in recent menopause, indicate that the ratio between vasodilator (NOx and prostacyclin) and vasoconstrictor (ET) bioavailability shifted towards the previous ones after O-ET, while it remained unchanged after T-ET; moreover, catecholamines levels were reduced by both treatments already from 1 month of therapy. These changes might represent very early beneficial effects evoked by ET on the cardiovascular system.

Usefulness of high-sensitivity IL-6 measurement for clinical characterization of patients with coronary artery disease.

Interleukin 6 (IL-6) may represent an early marker of inflammatory activation and may be useful to ameliorate risk stratification in patients with ischemic heart disease. The aim of this study was to verify the performance characteristics of an ultrasensitive immunoassay (Biosource International, Camarillo, CA) for high-sensitivity (hs)-IL-6 measurement in comparison with hs-R&D Systems (Abingdon, United Kingdom) and Immulite System (Diagnostic Products Corporation [DPC], Los Angeles, CA) methods in patients with ischemic heart disease. In addition, hs-C-reactive protein (hs-CRP) concentrations were measured, to evaluate the correlation with hs-IL-6 levels. We measured IL-6 and CRP serum levels in 39 patients with ischemic heart disease and in 12 controls. Out of the 39 patients studied, 13 were affected by unstable angina, 13 by post-acute myocardial infarction (AMI) unstable angina, and 13 by stable angina. The imprecision profile and functional sensitivity were performed measuring 9 different serum pools in 10 runs. The Biosource method had the best performance characteristics as compared to the others. Mean IL-6 level was higher in patients with unstable and post-AMI unstable angina with respect to controls. CRP levels were elevated in patients with post-AMI. In the whole population a high significant linear regression was observed between Biosource hs-IL-6 and hs-CRP serum levels. The Biosource method for IL-6 measurement is characterized by a high functional sensitivity that allows a better stratification of patients with ischemic heart disease.