Mechanism of interference by haemolysis in the cardiac troponin T immunoassay

Background The cardiac troponins have been shown to be sensitive and specific biochemical markers of myocardial infarction and highly prognostic for future adverse events in patients with acute coronary syndromes. There have been reports suggesting that haemolysis causes a negative interference in the cardiac troponin T (cTnT) assay but the mechanism(s) involved remain unknown. Here we show the effects of haemolysis and haemoglobin per se on the cTnT assay. Methods The effect of haemolysis was studied by the addition of prepared haemolysate to serum samples with known and clinically relevant cTnT levels. The effect of haemoglobin was studied by the addition of haemoglobin of increasing concentrations and noting its effect on the level of cTnT measured. The effect of putative proteases was determined indirectly by incubating samples with spiked cTnT with various protease inhibitors and observing the changes in the measured cTnT levels. Results The results show that both haemolysis, which is the release of haemoglobin and corpuscular contents, and haemoglobin itself negatively interfere in the cTnT assay in a concentration-dependent manner, although the former had a greater magnitude of effect. On haemolysis, indirect evidence suggests that proteases are released which degrade the cTnT in serum, thus causing the decreased levels detected. Pepstatin A, a reversible inhibitor of aspartic proteinases, effectively inhibited the loss of cTnT in serum at 37 degrees C and pH 7.4 over a 48-h period. We found that at a haemoglobin level of 0.75 g/L, cTnT declined by more than 10% of the initial concentration, suggesting that falsely decreased levels due to haemolysis may significantly affect the clinical utility of the assay. Conclusions Haemolysis, haemoglobin per se and possibly proteolysis play a role in the negative interference in cTnT assays. Measures to reduce this interference must be implemented.