by PD Dr Angelika Hammerer-Lercher

Measurement of cardiac-specific troponin I or T forms is part of the diagnostic pathway to rule in or rule out acute coronary syndromes, such as heart attack, as well as for prognostication of future cardiovascular disease. CLI chatted to Dr Angelika Hammerer-Lercher (Chief Physician and Director of Central Medical Laboratories, Central Medical Laboratories Feldkirch, Austria) to find out more about some of the limitations of these assays, particularly interference by macrotroponin.

What is cardiac troponin and how is it used as a biomarker of cardiovascular disease?

Cardiac troponins are a structural protein complex that is part of the thin filament of heart muscle cells and regulates the heart muscle contraction (Fig. 1). There are three isoforms of cardiac troponins: troponin I (TnI), troponin T (TnT) and troponin C (TnC). TnI inhibits the muscle contraction, TnT anchors the troponin complex as well as actin to tropomyosin on the thin filament. TnC binds calcium, which causes a conformational change in the troponin complex allowing myosin of the thick filament to bind to actin initiating the muscle contraction cycle. TnI and TnT are cardiac-specific isoforms whereas TnC is not, which is why we routinely measure the I and the T forms. Although most of the cardiac troponin complex is bound to the thin filament of heart muscle, there is also a very small unbound cytosolic pool of around 5%. In the event of heart muscle damage or ischemia, the cytosolic pool of free cardiac troponin is very rapidly released into the bloodstream, followed by release of the structurally bound troponins also. The absolute values of the troponins depend on the extent of the muscle damage: greater damage or ischemia results in higher and longer elevated levels of troponin. Troponin levels rapidly increase within a few hours after onset of symptoms and reach a plateau for around 5 to 14 days, with a slight difference between the I and T forms (the latter plateaus longer, in large infarcts even for up to 3 weeks), before declining to normal again. The troponins are, therefore, well-established markers for the diagnosis of acute coronary syndromes (ACS), such as heart attack, as well as being valuable biomarkers for prognosis of future cardiovascular disease like coronary artery disease heart failure or even stroke or hyper-tension. Clinicians, of course, have to use the cardiac troponin data in conjunction with the clinical symptoms and ECG data before proceeding in their clinical setting.

How is cardiac troponin concentration normally analysed?

Analysis of these biomarkers is normally done with cardiac-specific TnT and TnI immunoassays on analysers which are routinely used in laboratories. There are several high-sensitivity cardiac TnI (hs-cTnI) assays on the market, and just one for hs-cTnT (as the result of patent issues). In the recent years, the 0–1 hour and the 0–2 hour fast algorithms have been widely adapted (at least in Europe), which means that troponin is measured at presentation of the patient and then exactly 1 hour later or 2 hours later. The difference between the first and the second measurement (the delta value) is calculated, which allows the patient to be ruled out (has no ACS) or ruled in, or is in the middle the so-called grey zone. Decisions are not made on the troponin values alone, but also require the clinical suspicion and the ECG data, etc. There are also point-of-care assays, only a few of which are high-sensitivity assays at the moment but I believe more will be available soon. They may also be used for the fast algorithms if enough evidence of proper performance in this setting is given. However, it is important to understand that for both the routine assays in the laboratories and the point-of-care assays the delta values are assay-
specific and each of these assay methods provide different absolute values of troponin. Hence, the cut-off values are absolutely assay-specific an cannot be transferred from one assay to another. The cut-off and delta values are defined in the literature but they are only valid for the high-sensitivity assays that were investigated. The values are also stated in the “2023 ESC Guidelines for the management of acute coronary syndromes” and on the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) website also (https://shorturl.at/ADh7e).

Figure 1. Troponins form a complex that binds to actin and tropomyosin in cardiac muscle
(Adobe Stock.com)

The criteria for a high-sensitivity cardiac troponin assay are:

• Total imprecision (expressed as CV %) at the 99th percentile value should be ≤10% and the limit of detection should be below the 99th percentile.
• Values should be reported in ng/L.
• Assay should detect cardiac troponin in ∼50–95% of healthy individuals.

What are the limitations/challenges of cardiac troponin analysis?

Clinical suspicion: does the troponin concentration fit the context?
There are challenges with the troponin assays because they are highly sensitive, so elevated levels of troponin do not necessarily indicate a heart attack or ACS. There are chronic illnesses, such as heart failure or myocarditis, that can also cause troponin concentrations to increase above the cut-off or the upper reference level. However, these increased troponin concentrations are relatively stable values. The difference between acute and chronic disease is that for the diagnosis of an acute heart attack you must have a rise or fall of the troponin value and one of the measured troponin concentrations should be above the 99th percentile. Hence, it is very important to view the troponin measurements as part of the clinical situation along with the ECG or other imaging findings and then a proper diagnosis can be made. Additionally, it’s also important to be aware that in cases where the patient presents late, perhaps a few days after the acute event, the rise and fall of troponin concentrations will not be seen.

Pre-analytical problems
At the pre-analytical level, if the blood draw has been done in a fast, rough manner, hemolysis may have occurred, which in some assays may result in troponin measurements that are slightly lower with the risk of lower than the cut-off values. This might happen in rare cases of smaller heart attacks or early presenters. If an analyser does not have a hemolysis detection integrated, this circumstance can be difficult to be detected. Whenever ACS is clinically suspected, communication between clinicians and laboratorians is important to solve such an issue and a repeated measurement on a newly drawn blood sample is helpful.

Assay interference from heterophilic antibodies
The laboratory should also be aware of assay interferences, which for an immunoassay can occur from heterophilic antibodies. If heterophilic antibody assay interference is suspected, a dilution study should be done, as well as using heterophilic blocking tubes which block the heterophilic antibodies and result in a troponin measurement that has a lower value.

There are recommendations in the literature for how to proceed if assay interference is suspected [see Bibliography].

Macrotroponin interference
Macrotroponin is a complex of cardiac troponin and autoantibodies against cardiac troponin. Its presence is classed as an interference because it causes results from troponin assays that are higher than expected. For example, a viral infection could affect or stimulate the immune system, which would then produce autoantibodies, as well as anti-troponin autoantibodies. These can be produced after viral infections because we know that Epstein–Barr viruses cause, for example, the production of heterophilic antibodies. I don’t have proof, but I believe that cardiotropic viruses may also cause autoantibodies against the troponins. There are indeed case studies that found patients with unexpectedly high hs-cTn levels after COVID-19 infection due to macrotroponin and we observed the same in athletes [see Bibliography].

Normally, troponin is cleared from the blood by the kidneys and the half-life of troponin is around 2 hours. However, it can be imagined that if troponin is complexed with antibodies into a large molecule it is removed from the blood more slowly by the kidneys and, therefore, macrotroponin is present for longer than free troponin (possibly for several days or even weeks). Then if a patient presents at the Emergency Department with some symptoms suggestive of ACS and high troponin levels but no ECG findings, the clinician should be suspicious and communicate with the lab staff and ask whether this could be macrotroponin interference.

We observed unexpectedly high troponin levels with some athletes who came for post-COVID-19 check-ups to be cleared to train and compete. They had had symptoms typical of COVID-19, such as headache, cough, sore throat, but no new ECG changes. This raises suspicions about whether this athlete is healthy or not. With patients and athletes alike, further follow-up investigations have to be done, which are costly – imaging techniques, such as echocardiography, cardiac magnetic resonance imaging. These investigations could be avoided if an assay interference is found. One of the best and quickest ways to check such an interference is to use a different assay method. There was a big New Zealand study using residual plasma samples to demonstrate that mainly some hs-cTnI assay methods were affected by macro-troponin and to a lesser extend the hs-cTnT assay, so the measurement of hs-TnT may be of help if hs-cTnI is unexpectedly elevated. This can be done either with a collaborating lab or by a point-of-care device as most labs do not have two analysers themselves.

Additionally, an easy way to remove any macrotroponin that is present is to use a protein G Sepharose column which will bind and retain antibodies (which form part of the macrotroponin complex) so that the flow-through of this column would contain only free troponin and no macrotroponin resulting in lower troponin concentrations. The published cut-off level for this method is 40% (See Lam et al. in the Bibliography) with values below 40% hint that the original measurement was falsely high due to macrotroponin interference.

Are there any developments on the horizon that would improve cardiac troponin analysis?

The biotin interference was a big problem because a lot of nutrition supplements contain biotin; however, the manufacturer reacted and now this is no longer an issue. Macrotroponin interference is still a big challenge to cope with. I’m not aware of any developments that will prevent this at the moment. The main issue is to increase awareness of the potential for macrotroponin interference with troponin assays, particularly following COVID-19 or other viral infections. It is important that clinicians and lab staff think of the possibility of macrotroponin or other interferences if the troponin assay result does not tie in with the greater clinical picture and communicate with each other. Then the lab staff can investigate according to the IFCC recommendations.

The interviewee

Angelika Hammerer-Lercher MD, Chief Physician and Director of Central Medical Laboratories Central Medical Laboratories Feldkirch, Austria

Email: ahammerer-lercher@mzl.at

Bibliography
1. Definition of high-sensitivity cardiac troponin assay
1.a. Clerico A, Lippi G. The state-of-the-art of “high-sensitivity” immunoassay for measuring cardiac troponin I and T. J Lab Precis Med 2018;3:53 (https://jlpm.amegroups.org/article/view/4386/html).

2. Guideline for ACS including fast algorithm
2.a Byrne RA, Rossello X, Coughlan JJ et al. 2023 ESC Guidelines for the management of acute coronary syndromes. Eur Heart J Acute Cardiovasc Care 2024;13(1):55–161 (https://doi.org/10.1093/ehjacc/zuad107). Erratum in: Eur Heart J Acute Cardiovasc Care 2024;13(5):455  (https://doi.org/10.1093/ehjacc/zuad156).

3. Recommended laboratory work-up for assay interference
3.a Hammarsten O, Warner J V, Lam L et al. Antibody-mediated interferences affecting cardiac troponin assays: recommendations from the IFCC Committee on Clinical Applications of Cardiac Biomarkers. Clin Chem Lab Med 2023;61(8):1411–1419
(https://www.degruyter.com/document/doi/10.1515/cclm-2023-0028/html).
3.b Mair J, Giannitsis E, Mills NL et al. How to deal with unexpected cardiac troponin results. Eur Heart J Acute Cardiovasc Care 2022;11(4):e1–e3 (https://doi.org/10.1093/ehjacc/zuac023).

4. Recommended laboratory work-up for assay interference
4.a Hammarsten O, Becker C, Engberg AE. Methods for analyzing positive cardiac troponin assay interference. Clin Biochem 2023;116:24–30 (https://doi.org/10.1016/j.clinbiochem.2023.03.004).

5. Recommended laboratory work-up for assay interference
5.a Lam L, Aspin L, Heron RC, Ha L, Kyle C. Discrepancy between cardiac troponin assays due to endogenous antibodies. Clin Chem 2020; 66(3):445–454 (https://doi.org/10.1093/clinchem/hvz032).

6. Unexpectedly high levels of cardiac troponin in athletes
6.a Hammerer-Lercher A, Kissel C, Wittfooth S et al. A-019 Unexpectedly high cardiac troponin values in healthy athletes after SARS-CoV-2 infection. Clin Chem 2024;70(Suppl 1) (https://doi.org/10.1093/clinchem/hvae106.019).