Verification of the high-sensitivity troponin I assay method on Abbott Alinity I: Experience of the central Biochemistry Laboratory of Ibn Sina University Hospital in Rabat

 Corresponding author: Salima Lakrami Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Verification of the high-sensitivity troponin I assay method on Abbott Alinity I: Experience of the central Biochemistry Laboratory of Ibn Sina University Hospital in Rabat Salima Lakrami 1, 2, *, Loubna Zerrouki 1, 2, Mounya Bouabdellah 1, 2, Aziza M’hamdi Alaoui 1, 2, Imane Sekkal 1, 2 and Laila Benchekroun 1, 2 1 Central Biochemistry Laboratory, University hospital center Ibn Sina, Rabat, Morocco. 2 Mohamed V University, Faculty of Medicine and Pharmacy of Rabat, Morocco. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 358-368 Publication history: Received on 09 May 2025; revised on 15 June 2025; accepted on 17 June 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.22.3.0606 Abstract Verification of an analytical method in clinical laboratories is an essential process under the responsibility of biologists. It consists in assessing the analytical performance of a method and/or technique for assaying a biological parameter, in order to guarantee accurate and reliable results that benefit both the patient and the prescriber. The aim of our work, carried out in the Central Biochemistry Laboratory of the Ibn Sina University Hospital in Rabat-salé, is to verify the Troponin I (cTnI) assay « Range A » on Abbott's Alinity i automated system, using the chemiluminescent microparticle immunoassay (CMIA) method. The methodology adopted was based on the recommendations of the COFRAC (Comité Français d'Accréditation) accreditation technical guide, involving evaluation of the main analytical performances: repeatability, reproducibility and method comparison, plus an external quality assessment (EQA) to measure accuracy. The results obtained from this evaluation were compliant, and the CVs (coefficients of variation) produced were compliant and satisfactory with the supplier's data and with the Ricos learned society. Keywords: Troponin I; Method Verification; Analytical performance; External Quality Evaluation; Alinity I 1. Introduction Method verification in medical biology laboratories is a process that consists of evaluating the analytical performance of a method or technique for measuring a biological parameter, aiming to guarantee precise and reliable analytical results as well as the relevance of clinical interpretations beneficial to the patient and the prescriber. This is a permanent commitment of biologists and an essential step as stipulated in the quality standards, in particular the Guide for the Good Execution of Medical Biology Analyses (GBEA) applicable in Morocco since November 2011 to all laboratories, both private and public (1), ISO 15189 standards, learned societies (RICOS, SFCB, etc.) and the technical guide of COFRAC (French accreditation committee) (2,3,4). Troponin (Tn) is a structural protein of skeletal and cardiac muscle myofibrils. Troponin I (cTnI) measurement uses immunometric techniques with cardiospecific antibodies. It is the most sensitive and specific biomarker of myocardial damage. It currently represents the gold standard for early or retrospective diagnosis of myocardial infarction (MI) and the prognostic marker of unstable angina (5,6). The objective of our study is the verification of a "scope A" dosage method, involving a study of the analytical performance of Troponin I (cTnI) dosage on "Alinity i" Abbott, by a chemiluminescence microparticle immunological World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 358-368 359 method (CMIA), within the Central Biochemistry Laboratory of the Ibn Sina University Hospital of Rabat-Salé where the entire study was carried out, during a renewal of high-flow analytical equipment. 2. Material and methods 2.1. Biological principle of dosage The Abbott “Alinity i” Troponin I (cTnI) assay is based on a two-step immunological technique for the quantitative measurement of cTnI in human plasma and serum using chemiluminescence microparticle immunoassay (CMIA) technology. The sample and the paramagnetic microparticles coated with anti-troponin-I antibodies are brought together and incubated. Cardiac troponin-I present in the sample adheres to microparticles coated with anti-troponin-I antibodies. The mixture then undergoes a washing process. The acridinium-marked anti-troponin-I antibody conjugate is added to form a reaction mixture and then incubated. After another wash cycle, the preactivation and activation solutions are added. The resulting chemiluminescent reaction is measured in relative light units (URL). There is a direct relationship between the amount of Troponin present in the sample and the URLs detected by the optical system. 2.2. Dosing technique: The Troponin I assay was performed using a dedicated reagent kit (STAT High sensitive Troponin-I Reagent kit) on the immunology module of the Abbott Alinity i analyzer (code G73445R04, Lot 40058UD00-00193, Ref: 08P1332 / 08P1322) consisting of: • Microparticles: coated with anti-troponin-I antibodies (mouse, monoclonal) in TRIS buffer with a protein stabilizer (bovine). • Conjugate: Acridinium-marked anti-troponin-I antibodies (mouse/human chimeric monoclonal) in MES buffer with protein stabilizer (bovine) and human IgG. Data processing was carried out via Middleware EVM (BYG Informatique), serving as intermediary software between the Alinity platform and the biological validation software “Elabs” in order to obtain the different equations necessary for completing the verification file. The CVs (coefficient of variation) obtained were evaluated and compared to the CVs set by the learned society of RICOS (2014). 2.3. Description of the method Detailed description of the verification/validation method for Troponin I dosage, in accordance with document SH FORM 43 as part of its adaptation to the process approach of version 2022 of the ISO 15189 standard: Description of the method Analyte/Measurand Troponin-I Assay Principle of measurement Chemiluminescence microparticle immunoassay (CMIA). Method of measurement Alinity i STAT High Sensitive Troponin-I Reagent Kit (also called hsTnI Risk Strat) Primary sample type Serum or plasma Container type - Serum with and without separator. - Serum with thrombin-based clot activator. - Lithium heparin with and without separator. EDTA K2 EDTA K3 Sample pretreatment Transport to the laboratory: - Less than 8 hours: Store the primary tube at room temperature. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 358-368 360 - More than 24 hours: Store 2 ml of serum at 2 to 8°C. Centrifugation: - Serum samples: Centrifuge at 3000 to 3500 x g for 30 minutes. - Plasma samples: Centrifuge at 13 000 to 13 500 x g for 30 minutes. For frozen samples: - Thaw completely - Homogenize by vortexing at low speed or inverting 10 times. - Transfer the clarified sample to a sample cup. Unit - Default: ng/L - Other units: ng/mL, μg/L, pg/mL Interpretation criteria The measuring range of the Alinity i STAT High Sensitive Troponin-I assay is 10 to 50,000 pg/mL (0.01 to 50 ng/mL) Instrument Alinity i Reagent reference STAT High Sensitive Troponin-I Reagent Kit, REF 08P1327 /08P1337 Calibration material 08P1301 Alinity i STAT High Sensitive Troponin-I Calibrators Calibration type, number of levels and values 4 parameter logistic curve (4PLC, Y-weighted) 2.4. Analytical performance As part of method verification/validation, we have adopted strategy 1 or scope A (4), which consists of verifying the performance announced by the supplier or desired by the laboratory when implementing a new analysis method, in particular: reproducibility, repeatability, method comparison, accuracy. 2.4.1. Intermediate fidelity study (reproducibility) The reproducibility test is an essential step in the analytical performance evaluation process. It allows the same sample to be analyzed, in the same laboratory, on the same instrument, using the same method but with different environmental conditions: possible variation in the batch of reagents, in the calibration, by different operators (4). It allows the acceptance criteria for the differences observed between the results obtained and the pre-established compliance limits to be determined by comparing them with clinical requirements. In our study, the reproducibility assessment was carried out through the daily passage of three levels of Abbott® controls: low, medium and high. The reproducibility study was carried out on two different batches of reagents. The main objective was to evaluate the consistency and reliability of the test. The data allows the calculation of the mean, CV and standard deviation. The CVs calculated for each concentration level are compared with the CVs announced by the learned society of RICOS (2014). 2.4.2. Repeatability study Repeatability consists of dosing the same sample, by the same operator, under the same operating conditions, same environment, for all measurements, and for a short period. Repeatability study is an essential step when setting up a new automaton to know the initial performance of the system (reagent/instrument) and verify its correct functioning (4). In our study, the assessment of repeatability (intra-series imprecision) was carried out by measuring troponin I 20 times while respecting the above conditions, for the three control levels: low, medium and high. The data allows the calculation of the mean, CV and standard deviation. The CVs calculated for each concentration level are compared with the CVs announced by the learned society of RICOS (2014). World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 358-368 361 2.4.3. Method comparison study: Comparing an analysis method involves evaluating the results of patient samples (chosen to cover the pathophysiological extent) obtained by two different methods and highlighting differences that may affect the interpretation of the results. Our comparative study between Alinity ® and Architect i2000® was carried out on 22 patient samples, covering the entire measurement range (0.04 to 50 ng/ml). Each sample is processed in both machines. The results obtained by the old “Architect i2000” automaton are noted Xi and those of the new “Alinity i” automaton are noted Yi, thus allowing the determination of the linear regression line, the correlation coefficient and to draw the Bland and Altman diagram. 2.4.4. Accuracy The accuracy or closeness of agreement between the measured value and the true value of a measurand should be studied from a reference value obtained from a reference method. However, to date, medical biology laboratories evaluate their accuracy based on the results of external quality assessments (EQA) or the exchange of inter-laboratory results of samples (4). The Central Biochemistry Laboratory of the Ibn Sina University Hospital of Rabat-Salé is engaged in a BIO-RAD EQAS program (cycle 11) covering the period from 18 April 2022 to19 March 2023, allowing an External Quality Evaluation (EQE) of the dosage of cardiac markers, in particular Troponin I. The accuracy study corresponds to a comparison of the blind dosage results of Troponin I of the 12 unknown samples assayed in our laboratory during the study period compared to the «peer group» as well as «all techniques». 3. Results 3.1. Intermediate fidelity results (reproducibility) Table 1 Results of the Troponin-I reproducibility study on « Alinity i » Samples Number of values (N) mean (ng/ml) Standard deviation (ng/ml) CV (%) CV (%) Ricos CV (%) Ricos extended (K=1.418) Level 1 (low) 7 15.05 0.77 5.15 7.02 10.17 Level 2 (medium) 8 51.41 3.19 6.20 7.02 9.95 Level 3 (high) 8 907.20 16.38 1.80 7.02 9.95 The reproducibility results for all low, medium and high levels, giving coefficients of variation (CV1, CV2 and CV3) of 5.15%, 6.20% and 1.80% respectively (Table 1). These results were presented graphically using Levey-Jennings charts to further illustrate the results obtained (Figure 1, 2 and 3). World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 358-368 362 Figure 1 Representation of reproducibility results (low level) : Levey-Jennings curve and distribution around the mean Figure 2 Representation of reproducibility results (medium level): Levey-Jennings curve and distribution around the mean Figure 3 Representation of reproducibility results (high level) : Levey-Jennings curve and distribution around the mean 3.2. Repeatability results The results of the repeatability test for all low, medium and high levels, giving coefficients of variation (CV1, CV2 and CV3) of 3.02%, 2.49% and 1.65% respectively (Table 2). These results were presented graphically using Levey-Jennings charts to further illustrate the results obtained (Figure 4, 5 and 6). World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 358-368 363 Table 2 Results of the Troponin-I repeatability study on « Alinity i » Samples Number of values (N) mean (ng/ml) Standard deviation (ng/ml) CV (%) CV (%) Ricos CV (%) Ricos extended (K=1.260) Level 1 (low) 20 14.57 0.44 3.02 5.27 6.63 Level 2 (medium) 20 49.19 1.223 2.49 5.27 6.63 Level 3 (high) 20 886.14 14.61 1.65 5.27 6.63 Figure 4 Representation of repeatability results (low level) : Levey-Jennings curve and distribution around the mean Figure 5 Representation of repeatability results (medium level) : Levey-Jennings curve and distribution around the mean World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 358-368 364 Figure 6 Representation of repeatability results (high level) : Levey-Jennings curve and distribution around the mean 3.3. Method comparison results The results of the comparative study between Alinity® and Architect i2000® were analyzed by determining the linear regression line, defined by the equation Y = 0.94X - 0.17, with a correlation coefficient of 1.00 (Figure 7). In addition, the Bland and Altman concordance assessment method was performed, comparing the means of the measurements (abscissas) with their differences (ordinates) (Figure 8). Figure 7 Troponin I correlation study : Comparison between values obtained on Alinity® and Architect i2000® World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 358-368 365 Figure 8 Bland-Altman diagram 3.4. Accuracy results The results of the external quality assessment (EQA) are presented in table 3: Table 3 Results of the external quality assessment (*) : European Federation of Clinical Chemistry and Laboratory Medicine 4. Discussion Verification/validation of methods within a medical biology laboratory is a crucial and indispensable process that ensures accurate and reliable results. It is a regulatory requirement set out in GBEA and ISO 15189 (6,7). Within the framework of the NF EN ISO 15189 and 22870 standards, the medical biology laboratory must verify that its dosage methods are valid and mastered within the laboratory, and that they meet the needs of patients and prescribers. Sample Number (N) Lab Value (ng/l) Target (peer group) Target (all techniques) Bias (%) / (peer group) Bias (%) / (all techniques) Bias (%) optimal limit (EFLM)(*) Cycle 11 sample 1 78 2240 2234 2315 - 0.10 - 0.10 13.96 Cycle 11 sample 2 101 980 981 1017 - 0.10 - 0.10 Cycle 11 sample 3 109 54.1 56.2 58.4 - 3.72 - 7.34 Cycle 11 sample 4 102 232.19 254 266 - 8.73 - 12.80 Cycle 11 sample 5 107 2253.32 2283 2371 - 1.30 - 4.94 Cycle 11 sample 6 114 250.69 258 267 - 2.85 - 6.08 Cycle 11 sample 7 114 922.56 991 1028 - 6.89 - 10.30 Cycle 11 sample 8 117 2132.97 2267 2342 - 5.90 - 8.92 Cycle 11 sample 9 117 56.01 55.3 57.6 1.22 - 2.60 Cycle 11 sample 10 107 263.65 256 265 3.04 - 0.39 Cycle 11 sample 11 125 899.3 965 1018 - 8.71 - 11.60 Cycle 11 sample 12 121 56.45 54.2 56.9 4.11 - 0.87 World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 358-368 366 In our study, we carried out an on-site "scope A method verification" of the analytical performance of Troponin I Hs dosage on Alinity i from Abbott® during a renewal of analytical equipment at the Central Biochemistry Laboratory of Ibn-Sina University Hospital in Rabat-Salé. The most relevant modules of this verification were detailed in the results section of this work. A comparison of the results obtained with respect to the acceptable limits previously defined by learned societies (Ricos) and recently conducted studies on Troponin dosage allows for a critical reading and a correct interpretation of the clinical impact of the results. 4.1. Intermediate fidelity (reproducibility): The results of the study of the reproducibility of the Troponin I dosage on Alinity i are consistent and satisfactory with the supplier's data and the Ricos learned society (Table 4). Table 4 Results of the Troponin-I reproducibility study with comparison to RICOS data Samples Number of values (N) mean (ng/ml) Standard deviation (ng/ml) CV (%) CV (%) Ricos CV (%) Ricos extented (K=1.418) Conclusion Level 1 (low) 7 15.05 0.77 5.15 7.02 10.17 Compliant Level 2 (medium) 8 51.41 3.19 6.20 7.02 9.95 Compliant Level 3 (high) 8 907.20 16.38 1.80 7.02 9.95 Compliant 4.2. Repeatability The results of the repeatability study of the Troponin I assay on Alinity i are consistent with the supplier's data and the Ricos learned society (Table 5). The coefficients of variation (CV) obtained are satisfactory and appear to be consistent with those of the study mentioned by Westwood et al (8). Thus, it is concluded that the precision is consistent with the requirements set for the troponin I assay on Alinity i. Table 5 Results of the Troponin-I repeatability study with comparison to RICOS data Samples Number of values (N) mean (ng/ml) Standard deviation (ng/ml) CV (%) CV (%) Ricos CV (%) Ricos extented (K=1.260) Conclusion Level 1 (low) 20 14.57 0.44 3.02 5.27 6.63 Compliant Level 2 (medium) 20 49.19 1.223 2.49 5.27 6.63 Compliant Level 3 (high) 20 886.14 14.61 1.65 5.27 6.63 Compliant 4.3. Method comparison Comparison of assay results with the STAT Troponin-I kit on the Architect i2000® also showed better clinical precocity of the Abbott® technique, particularly for low values. The choice of antibodies has a significant impact on assay performance due to the presence of different forms of Troponin in the bloodstream. Regarding the detection limit, the literature reports a detection limit of 1.6 ng/L; which corresponds to the minimum concentration for which the coefficient of variation is less than 10%(9). 4.4. Accuracy Our laboratory is engaged in an External Quality Assessment (EQA) program aimed at improving laboratory performance. According to the study by Ricos et al (10), the results of the participating laboratories in this assessment are compared against a specification based on biological variation (BV), which was described in 1999 and was modified in 2019, when the European Federation of Laboratory Medicine (EFLM) database was published. Our accuracy results (Table 6) were consistent with the «peer group» as well as «all techniques».