American Association for Clinical Chemistry
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February 2011 Clinical Laboratory News: Here Come High-Sensitivity cTn Assays
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February 2011: Volume 37, Number 2


Here Come High-Sensitivity cTn Assays
Why Labs Need to Gear up Now

By Genna Rollins

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In the 2 decades since their introduction into clinical practice, the cardiac troponins I and T (cTn) have earned a solid place as markers of myocardial necrosis and for diagnosis and risk assessment in suspected acute coronary syndrome (ACS). Given the success of these markers, in vitro diagnostic (IVD) manufacturers have developed several generations of successively more sensitive assays, opening the door for expanded clinical indications for cTns. In recent years, these sensitive assays have detected cTn in patients with stable coronary artery disease, congestive heart failure, and chronic kidney disease, and have improved cardiovascular event risk prediction when considered in both traditional models and panels of biomarkers. Now, two studies using high-sensitivity cTn assays with limits of detection 10 times lower than conventional assays have reported not only finding cTn in community dwelling elderly patients without symptoms of heart failure and in a general population, but also associating rising cTn levels with risk of heart disease and death in the two groups.

These results herald a new avenue for risk assessment, diagnosis, and eventually, treatment of cardiac disease, but also will raise challenges for clinicians and laboratorians in understanding just what very low levels of circulating cTn mean, according to experts. “These high-sensitivity assays are going to be powerful tools for screening and potential early therapy to improve long-term outcomes,” said Fred Apple, PhD, professor of laboratory medicine and pathology at the University of Minnesota School of Medicine, and medical director of clinical laboratories at Hennepin County Medical Center in Minneapolis. “However, the clinical interpretation of what they mean in comparison to the assays we’re using today will need to be carefully reviewed and understood.”

Looking Below the Water Line

The two studies, published in the December 8, 2010 issue of the Journal of the American Medical Association, both used high-sensitivity cTnT assays and detected the analyte in sizable portions of the respective study populations that would not have been identified using cTn assays currently on the market in the U.S. One study took advantage of serial blood samples collected as part of the Cardiovascular Health Study, a multicenter prospective observational study of cardiovascular disease in older adults (JAMA 2010;304:2494–2502). The other accessed samples from the Dallas Heart Study, a large, multi-ethnic, population-based epidemiological study of Dallas County (Texas) residents (JAMA 2010;304:2503–12).

Out of nearly 5,900 people enrolled in the Cardiovascular Health Study, researchers accessed samples from 2,918 who did not have a prior diagnosis of heart failure and for whom there was adequate sample volume from baseline and follow-up visits. During a median follow-up of almost 12 years, they detected cTnT ≥3.00 pg/mL in nearly two-thirds of participants. The investigators also found a graded relationship between rising cTn levels and increased risk: participants with the highest cTn levels >12.94 pg/mL were more than twice as likely to develop heart failure in comparison to those with non-detectable cTn levels. Furthermore, in comparison to those with a 50% change or less, individuals with a >50% increase in cTn levels were at greater risk for both heart failure and cardiovascular death, with adjusted hazard ratios of 1.61 and 1.65, respectively. By the same token, participants with a >50% decrease in cTn values had a lower risk for heart failure and death, with adjusted hazard ratios of 0.73 and 0.71, respectively, in comparison to those with a 50% change or less.

“With this new high-sensitivity assay we were able to look down below the water line into the so-called normal ranges, where before the measurements were unreliable in that range,” said co-author Rob Christenson, PhD, professor of pathology and medical and research technology at the University of Maryland School of Medicine and director of core laboratories at the University of Maryland Medical Center in Baltimore. “We also observed that dynamic changes in levels are important. Individuals who had detectable high-sensitivity troponin levels initially were at greater risk of heart failure and cardiovascular death compared to patients who had undetectable levels. Among patients with detectable levels, a subsequent increase of more than 50 percent was associated with a greater risk for heart failure and cardiovascular death. Interestingly, if their high-sensitivity troponin levels decreased by 50 percent or more, then their risk also decreased.”

In the Dallas Heart Study, researchers used both standard and high-sensitivity cTnT assays to analyze samples from 3,546 participants who were age 30–65 when enrolled in the study between 2000–2002. They found detectable levels of cTn ≥0.003 ng/mL in one-quarter of subjects using the high-sensitivity assay, compared with just 0.7% with levels ≥0.01 ng/mL using the standard assay. The prevalence of cTn detected with the high-sensitivity assay differed considerably based on age, sex, and race. For example, 14% of participants 40–50 years had detectible levels of cTn, versus 57.6% of those 60–65 years. Men were three times more likely to have detectable levels than women, while one-third of African Americans had detectable levels in comparison to one-quarter of whites and one-fifth of Hispanics.

Using the high-sensitivity assay, the researchers also detected cTn in 16.2% of subjects without cardiovascular disease, chronic kidney disease, diabetes or hypertension, but found a rising prevalence of these conditions with increasing cTn levels. For instance, the prevalence of hypertension grew from 27.2% to 70.9% with rising cTn levels, and left ventricular hypertrophy rose from 7.5% to 48.1%. During a median follow-up of 6.4 years, the researchers found a rising incidence of all-cause mortality, a relationship that persisted even after adjustment for traditional risk factors and levels of other biomarkers, including C-reactive protein and N-terminal pro-brain-type natriuretic peptide.

“This test is among the most powerful predictors of death in the general population we’ve seen so far,” said James de Lemos, MD, associate professor of internal medicine at UT Southwestern Medical Center in Dallas, who was lead author of the Dallas Heart Study analysis and a co-author of the Cardiovascular Health Study report. “It appears that the higher your troponin T, the more likely you are to have problems with your heart, and the worse you’re going to do, regardless of other risk factors.”

What to Make of the Findings?

Researchers involved in the two studies and other experts agreed that the findings are important steps forward in identifying subclinical heart disease and cardiovascular disease risk, but much work remains in terms of understanding all their implications. “It’s still too early to say what role highly sensitive troponin testing will play in population screening. Although sensitive troponin levels are associated with cardiac structure, function, and prognosis, we haven’t really demonstrated that it will be useful in that setting. What we’d like to see is that high-sensitivity troponin analyses help decide therapy or interventions that subjects with high levels will benefit from,” explained Torbjorn Omland, MD, PhD, MPH, professor of medicine and deputy head of the Institute of Clinical Medicine at the Akershus University Hospital Campus of the University of Oslo in Norway. “These findings underscore that there are a number of factors associated with elevated troponin levels that don’t mean the patient has had myocardial infarction or acute coronary syndrome.” Omland was a co-author of the Dallas Heart Study analysis.

Omland’s latter point resonated with several researchers. “The notion that troponin T is released only through the process of myocardial necrosis, which is assumed to be the primary mechanism in the application of traditional troponin assays, seems not to fully explain our findings that two-thirds of asymptomatic individuals walking around in the community have this marker detectible in their sera,” said Stephen Seliger, MD, MS, assistant professor of medicine at the University of Maryland Medical Center in Baltimore and senior author of the Cardiovascular Health Study analysis. “There must be additional mechanisms that are causing the presence of this marker in circulation. Certainly a lot of research needs to be done to figure out what those mechanisms are.”

In a recent review article, Omland indicated that while researchers understand a great deal about cTn release, more remains to be discovered, including completely characterizing the cTn complex after onset of myocardial necrosis and determining whether cTn is released from the myocardium following reversible myocardial injury. Other scientists also emphasized the need for further research into the physiology and pathophysiology of cTn. “These are cardiac-specific troponins, and to the best of our current knowledge, a cell has to die for us to be able to measure cardiac troponin in the blood stream. Until or unless someone disproves that premise, release of troponins means there’s myocardial necrosis,” said L. Kristen Newby, MD, MHS, associate professor of medicine at Duke University School of Medicine and director of the biosignatures advanced biomarkers group at the Duke Clinical Research Institute in Durham, NC. “This release doesn’t say what caused the necrosis, but it says there’s necrosis.”

Newby also cautioned that as intriguing as the results of the two studies may be, for now, they raise more questions than answers. “The Holy Grail we’ve all been searching for is, if we find someone with an increasing, decreasing, or just a high level, how we should use this information to prevent them from having an adverse outcome, and what’s the time horizon for doing so? Do we need to intervene, and if so, do we intervene acutely, or do we have time to provide more conventional, primary and secondary interventions?” she said. “As these assays become increasingly sensitive we’ll have to deal with not only inter-but also intra-individual variability and the fact that cells eventually wear out and die, and what that means. If the assays become so sensitive that the majority of individuals are positive, they’ll become less useful as a prognostic marker, because the only time they’ll have value is when some people have detectible concentrations and others don’t or if there’s a gradation across levels of the biomarker and associated levels of risk.”

Current generation cTn assays reliably detect the steep rise in cTn levels that occur 2–6 hours after myocardial infarction (MI). However, the high-sensitivity assays used in these studies can detect much lower levels of ischemia, micronecrosis, and even normal levels (See Graph, below).

Detection Range of Different cTn Assays

Troponin figure

*Current generation assays detect rising cTn levels indicative of MI. High-sensitivity assays detect lower levels reflective of ischemia micronecrosis and even normal cell turnover.

Source: Reprinted from Am Heart J 2010;160:583–94, with permission from Elsevier.

A Rocky Implementation Predicted

As researchers continue to flesh-out the meaning of previously undetectable cTn values, experts urged laboratorians to prepare now for the time when high-sensitivity cTn assays become available commercially in the U.S. Several IVD manufacturers offer the test for research purposes here and are in the process of or plan to apply for Food and Drug Administration (FDA) clearance for the assays (See Table, below). However, these assays have been used in clinical settings in Europe for several years, where there have been challenges in interpreting their results. European-based clinicians cautioned their U.S. counterparts to expect some difficulties in implementing the assays.

“I’m absolutely sure users in the U.S. will suffer exactly the same confusion we have in Europe,” predicted Stefan Blankenberg, MD, professor of internal medicine and cardiology at Johannes Gutenberg University in Mainz, Germany. “We’ve had to learn to deal with this highly sensitive troponin assay for the diagnosis of acute coronary syndrome, because clinicians are confused now that we don’t have an arbitrary yes/no test. Rather, the troponin value is a continuous variable.”

The problem is this: guidelines on the Universal Definition of MI issued in 2007 by the European Society of Cardiology, ACC Foundation, American Heart Association, and World Heart Foundation called for a cTn measurement exceeding the 99th percentile of a normal reference population with a coefficient of variation ≤10% as an element of diagnosing MI, along with at least one additional criterion, such as symptoms or ECG changes indicative of ischemia. Those guidelines and NACB’s 2009 Laboratory Medicine Practice Guideline on Emerging Cardiovascular Risk Factors also note the importance of rising and/or falling cTn values in discerning MI, and call for measurements at the time of first assessment and 6–9 hours later to detect any pattern.

Already clinicians have had difficulty interpreting these guidelines using currently available assays to evaluate patients for suspected ACS in emergency settings. These challenges have been well-characterized, but essentially, clinicians have had a tendency to order cTn tests on patients with a low pre-test probability of ACS, leading to confusion about how to act on the results (CLN, April 2009). This issue will only be magnified with introduction of the new high-sensitivity assays, which will detect cTn values exceeding the 99th percentile in patients who have not had an MI. The lower detection limits of the new assays will provide diagnostic and prognostic information across a wide range of values, making passé the current paradigm of cTn being viewed like a qualitative positive-negative test. As one review paper put it, these assays “will increase the number of patients diagnosed with non ST-elevation MI that would previously have been labeled as unstable angina, and will allow an earlier detection of acute MI. However, a downside will be the detection of a wide range of pathologies with a significantly elevated cardiac troponin reflecting myocardial injury that is not related to ACS” (Am Heart J 2010;160:583–94).

Both Blankenberg and Omland have faced this dilemma. In an as yet unpublished analysis, Blankenberg proposes taking serial measurements at 0 and 3 hours with an initial value at the 99th percentile and a rise of 200–250% as a determinant of MI. Meanwhile, laboratorians in Norway have recommended not using the 99th percentile as the cutoff for MI, but rather a higher level of 0.03 µg/L. “This is to avoid over-diagnosis of MI, which is a particular concern in elderly patients, many of whom will have normal values higher than 0.014 µg/L circulating,” explained Omland.

Based upon data indicating that there are age and sex differences in low circulating cTn levels, others have questioned whether implementation of high-sensitivity assays in the workup of ACS warrants establishing different cutoffs for different populations or different times since symptom onset.

Comparison of Selected High-Sensitivity cTn Assays
 
LOD1,2
99th %ile2
10% CV2,3
Measurable values below 99th %ile
Beckman Coulter Access high sensitivity cTn I
0.0021
0.0086
0.0087
≥95%
Nanosphere high-sensitivity cTn I4
0.0002
0.0028
0.0005
75–95%
Roche high-sensitivity cTn T
0.002
0.013
0.012
≥95%
Singulex “molecular” cTn I4
0.0002
0.009
0.0009
≥95%

1Limit of Detection
2µg/L  
3Coefficient of Variation
4Experimental Assay

Source: Reprinted from Am Heart J 2010;160:583–94, with permission from Elsevier.

Gearing Up for Clinical Use

Only with further investigation will issues such as these be sorted out. In the meantime, researchers urged laboratorians to get up to speed on the literature related to high-sensitivity cTn assays. As these tests will almost certainly be adopted into clinical practice as soon as the FDA clears them, labs will do well to start planning for implementation now, according to Newby. “The clinical lab community needs to be engaged with developmental laboratorians in thinking ahead about how these assays should be used and what the downstream consequences are—for good or bad—before the assays are unleashed into hospitals,” she cautioned.

Laboratorians interested in beefing up their knowledge of high-sensitivity cTn assays will have several opportunities to do so in the near future. First, AACC is sponsoring a webinar on February 16 titled “hs - Troponin: Can It Play a Role in Risk-Stratifying and Managing Heart Failure Patients?” Christenson will be one of the faculty for this session. Second, Newby on April 5 will be discussing troponin elevations in the absence of overt ischemic heart disease as part of a joint AACC-American College of Cardiology session on the use of troponin in ischemic heart disease at the ACC Scientific Symposium in New Orleans. Finally, a new International Federation of Clinical Chemistry and Laboratory Medicine task force on clinical applications of cardiac biomarkers has formed with a goal of developing white papers to educate clinicians and laboratorians on the use of various cardiac biomarkers. Apple, who is a member of the task force, indicated that while the group has not settled on the biomarkers it will address, cardiac troponin would likely be high on the list.

Labs also will need to be prepared to educate a wide range of physicians about interpretation of the test, including some who may have very little experience with cTn. “These highly sensitive assays likely will bring new indications for troponin use, such that they won’t be used exclusively by cardiologists and emergency physicians who have focused on troponin in acute disease,” explained Christenson. “A primary care physician in the U.S. wouldn’t necessarily have dealt with troponin extensively because it’s used as an acute disease marker today. But these are the physicians who are caring for community-dwelling elderly, for example, and this test may be useful to them in the future, and they need to know about it.”

As the high-sensitivity assays roll out, collaboration between laboratorians and physicians will be critical, according to Apple. “When these assays are ready to be introduced into practice, laboratorians and clinicians need to understand that all troponin levels don’t equal MI. Clinicians will need to have the ability to interpret the troponin values based on patients’ clinical presentation. Never lose sight of that—clinical context will still be number one.”

For Further Information

  • Hochholzer W, Morrow DA, Giugliano RP. Novel biomarkers in cardiovascular disease: update 2010. Am Heart J 2010;160:583–94.
  • de Lemos JA, Drazner MH, Omland T, Ayers CR, et al. Association of troponin T detected with a highly sensitive assay and cardiac structure and mortality risk in the general population. JAMA 2010;304:2503–12.
  • de Filippi CR, de Lemos JA, Christenson RH, Gottdiener JS, et al. Association of serial measures of cardiac troponin T using a sensitive assay with incident heart failure and cardiovascular mortality in older adults. JAMA 2010;304:2494–2502.
  • Omland T. New features of troponin testing in different clinical settings. J Intern Med 2010;268:207–17.
  • Omland T, de Lemos JA, Sabatine MS, Christophi CA, et al. A sensitive cardiac troponin T assay in stable coronary artery disease. N Engl J Med 2009;361:2538–47.