Grossly Elevated False-Positive High-Sensitivity Troponin I Due to Heterophilic Antimouse IgG1 Antibodies

Although highly sensitive assays for the cardiac troponins are now considered the optimal biomarkers for the diagnosis of acute myocardial infarction, it is becoming increasingly apparent that there may be discordance between the 2 most common troponin markers. In the absence of myocardial injury, this poses a diagnostic problem for the laboratory and an even greater diagnostic clinical problem. Strategies are needed to rule out analytically false results. These may include dilution, treatment with blocking agents, precipitation, immunoglobulin removal, or gel filtration (1–4). In this case, a 77-year-old woman presented to the emergency department with a history of central chest pain, suspicious of acute coronary syndrome. She had grossly elevated troponin I (TnI; 1906 ng/L) on admission that increased over a period of 5 days to 2527 ng/L (reference interval <16 ng/L; Abbott Architect i2000SR). She was assigned a diagnosis of non–ST-segment elevation myocardial infarction and commenced antiplatelet therapy. It was noted, however, that a troponin T (TnT) concentration was 7 ng/L (reference interval <13 ng/L; Cobas E411), corresponding to the highest TnI elevation. Assay interference was suspected, and further investigations were undertaken on this latest sample. No other relevant laboratory tests were available. Polyethylene glycol (PEG) treatment by the addition of an equal volume of 25% aqueous PEG 6000 in PBS to the plasma sample normalized TnI (7 ng/L), with TnT unaffected. Comparison of protein A treatment to an inert gel control (Sepharose 2B) showed a 95% drop in TnI, with near complete immunoglobulin removal, as judged by protein electrophoresis. The specificity of protein A for the Fc region of immunoglobulins suggested that the interference was immunoglobulin mediated, and further investigation revealed the interference was not linear until dilution beyond 1:200. Consequently, we chose a plasma dilution of 1:400 in PBS to further investigate immunoglobulin-mediated interference. This was performed by incubation of the diluted plasma sample with purified isotype-specific mouse monoclonal antibodies to test whether they could correct the interference. These mouse immunoglobulin isotypes were separately purified by protein A chromatography of “in-house” monoclonal antibody supernatants. They comprised mouse immunoglobulin isotypes IgG1 κ, IgG2a κ, IgG2b κ, and IgM κ, which were derived from antibodies against osteocalcin, estrone-3-glucuronide, pregnanediol-3-glucuronide, and corticosteroid-binding globulin, respectively. Briefly we incubated the diluted patient sample with separate antibody test solutions, or mouse or sheep serum, overnight at 4°C in Eppendorf tubes followed by centrifugation for 10 min at 16 000 g. We then analyzed the supernatants for TnI. The final concentration of each test antibody solution was 200 µg/mL and for mouse and sheep serum 1.0 and 2.0 mg/mL respectively. Incubation of the sample with purified mouse IgG1 κ monoclonal antibody solution removed the interference and normalized TnI whereas TnI values following the other treatments were essentially unaffected when compared to the PBS control (Table 1). To confirm that incubation of the diluted sample with purified mouse immunoglobulin IgG1 κ removed the interference we subsequently tested 2 additional purified “in house” IgG1 κ mouse monoclonal antibodies. These mouse IgG1 κ antibodies were against cortisol and sex hormone-binding globulin and were also used 200 µg/mL with overnight incubation at 4°C with the 1:400 diluted patient sample. Treatment with these 2 other purified mouse IgG1 κ antibodies also removed the interference and showed recovery between 5% and 15%, similar to treatment of the sample with protein A, compared with >85% for all other treatments. TnI levels following treatment of plasma (1:400) with various reagents. Mouse IgG1 treatment-normalized TnI shown in bold. TnI levels following treatment of plasma (1:400) with various reagents. Mouse IgG1 treatment-normalized TnI shown in bold. One of the most frequent causes of false-positive results is the presence of heterophilic antibodies, which may be directed against animal immunoglobulins (5,). These are often alluded to by PEG precipitation or protein A treatment in which there is a drastic reduction in the percentage of recovery compared with unaffected samples. Compared with PEG precipitation, in which high-molecular-weight proteins are precipitated, protein A treatment is less intrusive and offers a high degree of specificity for the Fc region of immunoglobulins with their subsequent removal. This is highlighted in this case, where comparison by protein electrophoresis of the protein A–treated sample with the inert gel control showed immunoglobulin removal paralleling the removal of interference. Another hallmark of heterophilic antibody interference is the lack of linearity, which may resolve only at higher dilutions (2,). In our case, linearity was achieved only at dilutions exceeding 1:200, indicative of high-titer interfering antibodies. Therefore, we chose an even higher sample dilution (1:400) and used our readily available in-house protein A purified mouse monoclonal antibody library to determine the exact nature of that interference. Mitigation of interference by incubation with all 3 purified mouse IgG1 κ monoclonal antibodies and not by the other purified mouse monoclonal antibody isotypes and proteins defines the interference with the presence of heterophilic antimouse IgG1 κ antibodies. To our knowledge, this study is the first time such specificity has been reported. Interestingly, neither Scantibody nor diluted mouse serum treatment affected recovery, presumably masked by the very high titer of interfering antibodies, which have been reported to affect some assays (5). In conclusion, we used this reagent testing strategy and defined the interference in this case, which could theoretically be applied to other suspected false positives. TnI, troponin I; TnT, troponin T; PEG, polyethylene glycol. All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest: Employment or Leadership: J.G. Lewis, Canterbury Health Laboratories; H. Ploeg, Canterbury Health Laboratories. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research Funding: None declared. Expert Testimony: None declared. Patents: None declared.