Evaluation of High Sensitivity Cardiac Troponin I Assays

EVALUATION OF HIGH SENSITIVITY CARDIAC TROPONIN I ASSAYS

There are a number of cTnI assays on the market. These cTnI assays are not standardized at this time and studies have documented substantial differences across methods.(1) Apart from the lack of commutable reference material, other factors contributing to quantitative differences between the cTnI methods include the variable antibody immunoreactivity to different circulating cTnI forms and varying calibrators used in different cTnI assays.(1) The proper way to achieve complete standardization for cTnI assays would be to use antibodies with similar epitope specificities and a serum-based common reference material for calibration.(1) However, that is a complicated process and the progress has been slow.(2) In contrast to cTnI, there is only one manufacturer for the cTnT assay and the above shortcomings could be avoided.

The first-generation assay for cTnT used bovine cTnT as the reference material and exhibited non-specific binding to human skeletal muscle troponin,(3) but this problem was overcome by refinement of the detection antibody in the second-generation assay and the use of recombinant human cTnT for standardization in the third-generation assay.(4,5)The fourth-generation cTnT assay uses fragment antigen- binding (FAB) of two cTnT-specific mouse monoclonal antibodies in a sandwich format. The antibodies recognize epitopes located in the central part of the cTnT molecule (amino acid positions 125–131 and 135–147).

Detection of cTnT is based on an electrochemiluminescence immunoassay using a Tris(bipyridyl)-ruthenium(II) complex as a label.(6) The fourth-generation cTnT assay has a limit of detection (LoD) of 0.01 ng/mL, a 99th percentile cut-off point of 0.01 ng/mL, and a 10% coefficient of variation (CV) of 0.03 ng/mL.(6) For the diagnosis of acute myocardial infarction (AMI), the fourth-generation cTnT assay is considered the standard assay. In addition, samples in which there is an increases of cTn in the blood exceeding the 99th percentile of the normal reference population, the guidelines suggest that the CV of the ideal cTn assay used is ≤ 10% at the 99th percentile concentration.(7,8) Clearly, the fourth-generation cTnT assay lacks adequate precision. The new high-sensitive cTnT (hs-cTnT) assay is a modification of the fourth-generation cTnT assay.(6) The biotinylated capture antibody was not changed. The detection antibody was genetically re-engineered, replacing the constant C1 region in the monoclonal mouse FAB fragment with a human IgG C1 region, leading to a mouse-human chimeric detection antibody.(6) The rationale for this replacement was to further reduce the susceptibility to interference by heterophilic antibodies. The variable region of the detection antibody is identical to that of the fourth-generation assay. The analytical sensitivity was improved by increasing the sample volume from 15 μL to 50 μL, increasing the ruthenium concentration of the detection antibody, and lowering the background signal via buffer optimization.

As a result of these modifications, the analytic performance of the hs-cTnT assay was significantly improved; specifically, the LoD was 0.003 ng/mL (3 ng/L), the 99th percentile cut-off point was 0.014 ng/mL (14 ng/L), and the CV was 10% at 0.013 ng/mL (13 ng/L).(6) Due to a lower LoD and a increased precision, the hs-cTnT assay is able to detect more subtle elevations indicative of cardiac Injury

Recommendations for the use of cardiac troponin (cTn) measurement in acute cardiac care have recently been published. Subsequently, a high-sensitivity (hs) cTn T assay was introduced into routine clinical practice. This assay, as others, called highly sensitive, permits measurement of cTn concentrations in significant numbers of apparently illness-free individuals. These assays can measure cTn in the single digit range of nanograms per litre (=picograms per millilitre) and some research assays even allow detection of concentrations <1 ng/L.Thus, they provide a more precise calculation of the 99th percentile of cTn concentration in reference subjects (the recommended upper reference limit [URL]). These assays measure the URL with a coefficient of variation (CV) <10%. The high precision of hs-cTn assays increases their ability to determine small differences in cTn over time. Many assays currently in use have a CV >10% at the 99th percentile URL limiting that ability. However, the less precise cTn assays do not cause clinically relevant false-positive diagnosis of acute myocardial infarction (AMI) and a CV <20% at the 99th percentile URL is still considered acceptable.

ANALYTIC ISSUES

The minor analytic issues that occur with all immunoassays will be much more critical with these very high-sensitivity assays, in which minor changes could make marked differences. For example, cTnT is reduced by hemolysis(10) and some cTnI assay values are increased. Given that most samples from critically-ill patients are obtained from lines, careful scrutiny of the important preanalytical processes used to obtain samples will be critical. In addition, the recent problem with the hs-cTnT calibrator, involving a drop in the percentage of detectable values from more than 50% in the initial assay lots to 25% in some of the lots, illustrates that some degree of local quality assurance of values near the 99th percentile URL will be essential.

USING HIGH-SENSITIVITY CARDIAC TROPONIN TO DIAGNOSE ACUTE MYOCARDIAL INFARCTION

Because high-sensitivity assays detect values in apparently healthy participants with subclinical cardiac disease, a significant proportion of patients presenting with possible AMI will have hs-cTn values above the 99th percentile URL. Thus, all AMI guidelines recommend serial cTn sampling to observe a rise and/or fall in values in a clinical setting, giving rise to significant suspicion for an acute coronary syndrome. Unfortunately, a clear definition, based on data, of the optimal significant rise/fall in serially analyzed cTn concentrations is lacking. One approach to this problem has been to measure so called biological variation, which is the change that might be present due to conjoint analytic and biological variation and to develop a value (the reference change value [RCV]) above which one could be sure that spontaneous variation has been exceeded. Because contemporary cTn assays measure so few healthy individuals, it was previously impossible to calculate this value. The current hs-cTn assays correct this problem. Serial cTn changes in a patient can be attributed to pathological causes when the value is higher than the RCV calculated in healthy persons; similarly, a serial change higher than the RCV observed in persons in a chronic, stable condition will indicate the existence of an ongoing acute event. Caution is required when interpreting the RCV. First, RCV values are dependent on the method used to measure cTn and, second, the RCV used to evaluate a rising cTn pattern can differ from that used to evaluate a falling pattern. A recent report has summarized the RCV observed for the hs-cTn assays that are currently in use or close to being marketed. For hs-cTn, the RCV for evaluating short-term (hours) rising patterns varied from 26% to 90%, whereas the data for falling patterns varied from  -21% to  -47%. For different hs-cTnI assays, the reported values were from 46% to 69% for rising kinetics and from -16% to  -41% for falling ones. For all the evaluated hs-cTn assays, higher changes are required for rising values than for falling values. However, it is now clear from clinical data that values lower than the RCV will be needed to optimize the sensitivity of hs-cTn and that many patients without an acute event may have values that exceed the RCV. Thus, there will be a trade-off between sensitivity and specificity in terms of the definition of an appropriate delta for clinical use (11) Defining an optimal delta value for clinical use is complex. Unfortunately, there are only a few articles that could be taken to provide definitive data in this area. Some groups have advocated the use of relative change criteria, such as a 50% change in values, based on consideration of the RCV.(12) Others have argued that absolute changes perform more robustly.(13) Reflection on these efforts gives rise to several important principles:

ü  Consistent timing is obligatory to compare the performance of various metrics.

ü  Each assay must be evaluated separately.

ü  Data based on a 6-h evaluation cannot be inferred to work at 1 or 2 h based on dividing by the time interval. Such an approach suggests that cTn release is continuous, which is not the case in many situations. In addition, the small amount of change that such an approach demands of the assays is not feasible, given the intrinsic imprecision of the approach.

ü  A proper gold standard is key to proper use. If diagnosis of AMI is based on less sensitive assays, the degree of change will be greater than if the hs-cTn assay is used as its own gold standard, because smaller infarctions will now also be included.(14)

ü  Small changes can make large differences, and therefore, as indicated above, quality control of the assays is essential.

ü  Since cTn release is perfusion-dependent, clinicians need to be aware that ‘‘open artery’’ AMIs may provide a different signal than those that occur behind a total occlusion.

ü  It is already clear that absolute changes or a reduction in the expected percent change12 will be needed if the initial hs-cTn value is significantly elevated.

ü  Emergency departments and cardiology services will need to consider whether they wish to set criteria to enhance sensitivity or specificity.

ü  A rising and/or falling pattern is not specific for AMI; only for acute events. Thus, sepsis and pulmonary embolism, etc, can cause such a pattern of elevations.

THE CONTRIBUTION OF HIGH-SENSITIVITY CARDIAC TROPONIN ASSAYS TO THE MANAGEMENT OF CHRONIC CARDIOVASCULAR DISEASE

The management of chronic cardiovascular disease will be a major area of improvement in patient care. It is now clear that minor elevations of hs-cTn are common and are almost always associated with cardiovascular comorbidities.(15) Some of these comorbidities are so subtle that they are not detectable clinically or even with imaging studies. However, in a variety of community-based studies, these comorbidities have been shown to be associated with an increased risk for adverse cardiac events over time. Thus, in future, we may well be measuring hs-cTn to detect the subtle development of cardiovascular comorbidities; such detection could help to develop strategies that can be applied at an early stage in the hope of preventing the development of cardiovascular events. DeFillippi et al.(16) have provided an example of this in a surveillance study that used hs-cTnT to detect older individuals at risk for the development of heart failure. Not surprisingly, individuals with the more elevated values were at higher risk but, in addition, those who showed an increase in an interim blood sample were also at greater risk. Could interventions have prevented disease? Our task is to discover the answer to that question. This same principle applies to patients with congestive heart failure, in which the potent prognostic effects of hs-cTn have been demonstrated both in patients with acute and in those with chronic heart failure. Again, as in the community cohorts, a rising pattern of values over time was associated with increased risk. Because there is much less variation in hs-cTn than in brain natriuretic peptide, some authors have even suggested that it might be a better marker to use to titer therapy. The above-mentioned considerations are merely examples of some of the data that have accumulated in this important area. New data have emerged in hypertrophic cardiomyopathy and in the prediction of postoperative AMI. Nascent areas of inquiry, such as the use of hs-cTn to monitor drug toxicity, will likely benefit from the emergence of new data as well.

CONCLUSIONS

High-sensitivity assays are currently available. If we use these assays optimally, they will represent a major advance. If we fail to understand how to use them, they will become a source of confusion and a common cause of medical error. Hopefully, this article will help those who are ready and willing to move forward.

REFERNCES

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