High incidence of macrotroponin I with a high-sensitivity troponin I assay

Abstract

Background:Cardiac troponin is the preferred biomarker of myocardial injury. High-sensitivity troponin assays allow measurement of very low levels of troponin with excellent precision. After the introduction of a high-sensitivity troponin I assay the laboratory began to receive enquiries from clinicians about clinically discordant elevated troponin I results. This led to a systematic investigation and characterisation of the cause.Methods:Routine clinical samples were measured by the Architect High Sensitive Troponin-I (hsTnI) and the VITROS Troponin I ES assays (VitrosTnI). Results that were elevated according to the Architect but not the VITROS assay (Group 1) or results elevated by both assays but disproportionately higher on the Architect (Group 2) were re-analysed for hsTnI after re-centrifugation, multiple dilutions, incubation with heterophilic blocking reagents, polyethylene glycol (PEG) precipitation, and Protein A/G/L treatment. Sephacryl S-300 HR gel filtration chromatography (GFC) was performed on selected specimens.Results:A high molecular weight complex containing immunoreactive troponin I and immunoglobulin (macrotroponin I) was identified in 5% of patients with elevated hsTnI. Patients with both macrotroponin and myocardial injury had higher and longer elevation of hsTnI compared with VitrosTnI with peaks of both macrotroponin and free troponin I-C complex on GFC.Conclusions:Circulating macrotroponin I (macroTnI) causes elevated hsTnI results with the Architect High Sensitive Troponin-I assay with the potential to be clinically misleading. The assay involved in this investigation may not be the only assay affected by macrotroponin. It is important for laboratories and clinicians to be aware of and develop processes to identify and manage specimens with elevated results due to macrotroponin.

Laboratory-related issues in the measurement of cardiac troponins with highly sensitive assays

“…False-positive hs-cTn results may also be attributed to the presence of complexes between antibodies and cTn, resulting in a delayed clearance of the macrocomplex from the circulation. Initially recognized by Plebani et al [73], the presence of macrotroponin I was identified in 5% of patients with elevated hs-cTn I values, when measured with the Abbott Architect assay; but given the biological characteristics of the interferent, the investigated assay may not be the only hs-cTn I assay affected by this problem [74]. A case of macrotroponin T has been also reported [75].…”

The 99th percentile of reference population for cTnI and cTnT assay: methodology, pathophysiology and clinical implications

“…It is well known that interferences from, for example troponin macro-aggregate [70,71] and heterophilic [72] or auto-antibodies [73], may produce aberrant results in troponin testing in individual samples. Finally, the EP28-A3c IFCC-CLSI document [66] recommends that all pre-analytical factors (including subjects preparation, sample collection and processing), the analytical method procedure (including lot-to-lot variation of materials and calibrators) and instrumentation must be carefully defined and used for testing the reference individuals in order to minimize the number of aberrant results.…”

The investigation of interferences in immunoassay

“…It appears that the autoantibodies are directed against cTnI but only when it is in the circulating cTnI-cTnC-cTnT (I-T-C) complex [29]. The prevalence of circulating antibodies to cTnI have been described as between 5% [30] and 12.5% [31]. This is much higher than seen in routine clinical practice and implies we are missing a substantial number of cases.…”

“…The general principles of PEG precipitation of macro-complexes can be applied to any smallish molecule bound to an immunoglobulin. There have been reports of macro-TSH being investigated with PEG precipitation [16,54] and also troponin [30,55]. However, the behaviour of any assay used in conjunction with PEG precipitation needs to be carefully validated before use.…”

“…Gel filtration column chromatography using an agent such as Sephacryl 300 which has a separation range of approximately 10,000 -1,500,000 daltons MW enables the molecular weight associated with the analyte to be defined [30].…”