NEW TROPONIN I STANDARDS
for AMI Diagnosis

Introduction
The acute coronary syndromes represent a continuum of ischemic heart disease ranging from unstable angina to myocardial infarction with large areas of cardiac necrosis. They account for approximately 7 million deaths annually in the world.¹ Manifestations of an acute myocardial infarction (AMI) include sweating, breathlessness and intense chest pain, but symptoms can be far milder, more varied and potentially misdiagnosed, especially in women.² The recent introduction of more specific cardiac biomarkers such as the cardiac troponins have contributed to largely modify our current practice not only with respect to diagnosis and risk assessment, but also to therapeutic decision making in patients with suspected acute coronary syndromes. The IMMULITE^® 2500 STAT Troponin I assay is one of the newest assays designed to meet the clinical guidelines for acute myocardial infarction recommended by the European Society for Cardiology (ESC), the American College of Cardiology (ACC) and the National Academy for Clinical Biochemistry (NACB). Our evaluation of this product demonstrates that it is a rapid, sensitive troponin assay that meets the ESC/ACC and NACB criteria as closely as, or better than, other commercially available assays.

From the WHO to the ESC/ACC criteria for diagnosis of AMI
AMI corresponds to a loss of cardiac myocytes (necrosis) as a consequence of prolonged ischemia. In the late 1970s, the World Health Organization (WHO) proposed a definition of AMI based on the presentation of at least two of three specific criteria (Table 1), which have now been reexamined.

Table 1. ESC/ACC vs. WHO definition of AMI.

click image to enlarge

Prior to the late 1990s, markers such as myoglobin and creatine kinase MB (CK-MB) were used to assess cardiac damage and to diagnose AMI; these markers are not specific to cardiac myocytes, however, and can also appear in serum as a result of skeletal muscle injury. The advent of sensitive and specific cardiac biomarkers—namely, cardiac troponins I (cTnI) and T (cTnT)—has provided clinicians with completely specific cardiac markers capable of detecting minor myocardial injury. They have also been recognized as useful not only for emergency room triage of patients experiencing an episode of acute chest pain as reported by Hamm, et al.,³ but also to predict the risk of mortality in patients with acute coronary syndromes.^4,5,6 As early as 1996, Antman et al.⁷ linked the release of cardiac troponin to an increased risk of subsequent adverse coronary events, clearly demonstrating that small myocardial injury might be detectable.

This and other evidence prompted the ESC/ACC to publish a joint document in 2000, redefining AMI (Table 1) according to the concept that "any amount of myocardial necrosis caused by ischemia should be labelled as an infarct" and recommending cardiac troponins as the markers of choice.⁸ It should be noted that just a year prior to the ESC/ACC position statement, the NACB, using the WHO criteria, recommended that two decision limits be used rather than one, with a lower cutoff (97.5 percent) being considered the threshhold for making a diagnosis of cardiac injury as distinguished from AMI, which could be diagnosed only at a higher value determined by an assay's ROC cutoff.⁹ However, the 2004 NACB guidelines no longer support this dual-cutoff model and have adopted criteria very similar to those of the ESC/ACC. The NACB also recommends that laboratories "should perform STAT cardiac marker testing . . . with a target turnaround time (TAT) of one hour or less" (i.e., from the time of blood collection to the reporting of results).¹⁰ The evolution of these criteria is illustrated in Figure 1.

Figure 1. Evolution of the AMI decision limit (cutoff value).

Cardiac troponin in the context of the ESC/ACC criteria
The reference interval for troponin assays, as defined by the ESC/ACC, is the 99th percentile of a normal (i.e., healthy) population and is considered the cutoff value for the diagnosis of AMI. This value should be measurable with acceptable precision, i.e., the coefficient of variation (CV) ideally less than or equal to 10 percent. In this regard, the Committee on Standardization of Markers of Cardiac Damage (CSMCD) of the International Federation of Clinical Chemistry (IFCC) recently published data showing that, of 13 commercial assays, none was able to achieve a 10% CV at its 99th percentile cutoff.¹¹ In general, manufacturers are striving to improve the assays around the 99th percentile value to avoid potentially false-positive analytical results.¹⁰

Complete necrosis of myocardial cells following an infarction is not immediate and requires at least 4 to 6 hours; this results in a 3- to 4-hour delay in the appearance of troponin in the blood after the onset of chest pain. Consequently, the level observed at the admission of the patient may be less than the baseline concentration at the 10% CV. Levels should increase above this value, however, over the initial 24 hours after the occurrence of an AMI¹⁰ and remain high for up to 10 to 14 days. Thus, serial samples after admission (i.e., at 6 to 9 hours and 12 to 24 hours) are recommended. Figure 2 presents a typical time course for the release of cTnI following ischemic cardiac injury.

Figure 2. Representative release of cTnI into serum.

The increase in cardiac troponin can occur in settings other than acute ischemia (spontaneous ischemia or percutaneous coronary intervention) and, in such cases, clinicians may have to search for another etiology for the elevation (Table 2).

Table 2. Non-AMI-related causes of increase in cardiac troponin.12,13

Our experience with the new IMMULITE 2500 STAT Troponin I assay
DPC's IMMULITE 2500 STAT Troponin I assay is a solid-phase, two-site chemiluminescent immunometric assay that uses monoclonal murine antibody immobilized on beads and goat polyclonal antibody labeled with alkaline phosphatase as the tracer. As in DPC's earlier assays, both antibodies recognize epitopes localized in the stable N-terminal region of the protein (residues 33 to 110). The different cTnI complexes and isoforms^14,15 are also recognized in almost equal proportions, at least for the tested complexes as described by Wu et al.¹⁵ The reagents have been formulated to minimize the interference of factors such as heterophilic antibodies, human antimouse antibodies (HAMA)¹⁶ and rheumatoid factor.¹⁷ Additionally, the incubation cycle of this new assay has been reduced to 10 minutes (as compared to the 30 minutes required by previous assays) and the results are obtainable in 15 minutes, more than meeting the NACB TAT recommendations mentioned earlier.

The analytical sensitivity stated in the package insert is 0.10 µg/L. To identify the lowest concentration associated with a 10% CV, we generated a precision profile in the low range of concentrations, analyzing eight pools of serum samples in duplicate once a day over 20 days (one reagent lot and three calibrations at days 1, 11 and 16). We obtained a 10% CV at 0.29 µg/L (Figure 3)—very close to the value measured from the precision data in the package insert (which used heparinized samples).

Figure 3. Precision profile of the DPC assay with serum.

To determine the 99th percentile, we analyzed samples obtained from 504 blood donors (healthy population; 285 males and 219 females; aged 18–64 years). Serum samples were stored at –20°C until cTnI measurement, which was performed between the 45th and the 120th day of storage. For the study, the low cTnI concentrations (< 0.10 µg/L) were recalculated from the counts per second (CPS). As shown in Figure 4, 492 samples (97.6%) had a cTnI concentration less than or equal to 0.10 µg/L. The calculated median value was 0.02 µg/L and the 99th percentile was 0.16 µg/L.

Figure 4. cTnI concentration distribution.

According to Panteghini et al.,¹¹ the quality of a cTnI assay is reflected in the 10% CV / 99th percentile ratio. If this ratio is around 2 or less, the assay is regarded as "good." In the case of the IMMULITE 2500 STAT Troponin I assay, this ratio (0.29 µg/L / 0.16 µg/L) is less than 2 (1.81), establishing its quality and proposing a rounded 10% CV cutoff value of 0.30 µg/L.

Conclusion
The performance of this new assay improves the detection of low cTnI concentrations despite a shortened incubation period. Thus, the assay clearly conforms to the new criteria defined by the ESC/ACC as well as the NACB guidelines, although the coefficient of variation at the 99th percentile is slightly higher than 10% (around 15%).

Editor's note: A fully detailed DPC technical report titled IMMULITE^® 2500 STAT Troponin I: Meeting the New Standards for AMI Diagnosis (catalog number: ZB235) is now available to international customers. Please contact your DPC representative to request your copy.