Standardisation of Troponin I Measurements: an Update

Standardization of cardiac troponin I (cTnI) measurement is important because of the central role for diagnosos of myocardial infarction. In blood, cTnI is present as a heterogeneous mixture of differnent molecular species. The analytical problem caused by this microheterogeneity may be circumvented by recognition of a unique, invariant part of the molecule that is common to all components of the mixture. Antibodies used for development of cTnI assays should selectively recognize epitopes within this invariant part, leading to a consequential increase on the homogeneity of immonuassay reactivity. This should be associated with the use of a reference material (RM) that represents the natural and major antigen in blood after tissue release, i.e. the troponin complex. Although a primary RM for cTnI is available, studies indicate that cTnI assays remain without harmony after recalibration using this material. In order to achieve closer comparability of cTnI values between assays, the use of a secondary RM, consisting of a panel of human serum pools, is proposed for use by manufacturers to calibrate their assays. To assign true cTnI concentration values to this secondary RM, establishment of a reference measurement procedure for cTnI is required. A practical approach to the development of a reference procedure could be to design an immunochemical assay with well-characterized specificity to the invariant part of the cTnI molecule and calibrated using the primary RM.JRC.D.2-Reference material

Evaluation of standardization capability of current cardiac troponin I assays by a correlation study: results of an IFCC pilot project

Background: As a part of an International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) project to prepare a commutable reference material for cardiac troponin I (cTnI), a pilot study evaluated current cTnI assays for measurement equivalence and their standardization capability. Methods: cTnI-positive samples collected from 90 patients with suspected acute myocardial infarction were assessed for method comparison by 16 cTnI commercial assays according to predefined testing protocols. Seven serum pools prepared from these samples were also assessed. Results: Each assay was assessed against median cTnI concentrations measured by 16 cTnI assays using Passing-Bablok regression analysis of 79 patient samples with values above each assay’s declared detection limit. We observed a 10-fold difference in cTnI concentrations for lowest to highest measurement results. After mathematical recalibration of assays, the between-assay variation for patient samples reduced on average from 40% to 22% at low cTnI concentration, 37%–20% at medium concentration, and 29%–14% at high concentration. The average reduction for pools was larger at 16%, 13% and 7% for low, medium and high cTnI concentrations, respectively. Overall, assays demonstrated negligible bias after recalibration (y-intercept: –1.4 to 0.3 ng/L); however, a few samples showed substantial positive and/or negative differences for individual cTnI assays. Conclusions: All of the 16 commercial cTnI assays evaluated in the study demonstrated a significantly higher degree of measurement equivalence after mathematical recalibration, indicating that measurement harmonization or standardization would be effective at reducing inter-assay bias. Pooled sera behaved similarly to individual samples in most assays.JRC.D.2-Standards for Innovation and sustainable Developmen

Standardisation of Cardiac Troponin I Measurement: Past and Present

The laboratory measurement of cardiac troponin (cTn) concentration is a critical tool in the diagnosis of acute myocardial infarction (MI). Current cTnI assays produce different absolute troponin numbers and use different clinical cut-off values; hence cTnI values cannot be interchanged, with consequent confusion for clinicians. A recent Australian study compared patient results for seven cTnI assays and showed that between-method variation was approximately 2- to 5-fold. A major reason for poor method agreement is the lack of a suitable common reference material for the calibration of cTnI assays by manufacturers. Purified complexed troponin material lacks adequate commutability for all assays; hence a serum-based secondary reference material is required for cTnI with value assignment by a higher order reference measurement procedure. There is considerable debate about how best to achieve comparability of results for heterogeneous analytes such as cTnI, whether it should be via the harmonisation or the standardisation process. Whereas harmonisation depends upon consensus value assignment and uses those commercial methods which give the closest agreement at the time, standardisation comes closer to the true value through a reference measurement system that is based upon long-term calibration traceability. The current paper describes standardisation efforts by the International Federation of Clinical Chemistry and Laboratory Medicine Working Group on Standardization of cTnI (IFCC WG-TNI) to establish a reference immunoassay measurement procedure for cTnI of a higher order than current commercial immunoassay methods and a commutable secondary reference material for cTnI to which companies can reference their calibration materials.JRC.D.2-Reference material

Development of a Candidate Secondary Reference Procedure (Immunoassay Based Measurement Procedure of Higher Metrological Order) for Cardiac Troponin I: I. Antibody Characterization and Preliminary Validation

In this study, the first steps in the development of a secondary reference measurement procedure (RMP) "higher metrological order measurement procedure" to support the cardiac troponin I (cTnI) standardization initiative is described. The RMP should be used to assign values to serumbased secondary reference materials (RMs) without analytical artifacts causing bias. A multiplexed bead-based assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to identify the optimum monoclonal antibody pair (clones 560 and 19C7) for the RMP. Using these antibodies, an ELISA-based procedure was developed to accurately measure the main cTnI forms present in blood. The proposed RMP appears to show no bias when tested on samples containing various troponin complexes, phosphorylated and dephosphorylated forms, and heparin. The candidate assay displayed suitable linearity and sensitivity (limit of detection, 0.052 mg/L) for the measurement of the proposed cTnI secondary RMs. Preliminary comparison data on patient samples with a commercial cTnI assay are also provided to support the suitability of RMP for value assignment to RMs. Full validation and final assessment of the RMP will be performed through transferability and intercomparison studies.JRC.D.2-Reference material