CDU Contributing Editor

PHILADELPHIA, Pennsylvania New cardiac markers such as troponin I, troponin T and, most recently, B-type natriuretic peptide (BNP) and proBNP, have revolutionized the diagnosis of myocardial infarction and heart failure in the emergency department. Troponin testing is now a key component of the process for diagnosing heart attack, and troponin levels are also providing important information to physicians for assessing patient prognosis following an acute coronary event. As shown in Table 1, sales of cardiac marker products for the leading supplier of point-of-care (POC) cardiac marker testing systems, Biosite Diagnostics (San Diego, California), have increased at a compound annual rate of over 100% over the past three years. Total sales of cardiac marker tests are considerably higher than the figures shown in Table 1 when testing performed in all sites including the central laboratory is included, and are estimated at $107 million in the U.S. for 2002 excluding sales of BNP test kits.

As another indicator of the trend in cardiac marker testing, troponin tests performed in the U.S. Medicare population increased almost 17-fold from 1997 to 2002. Markers such as cardiac troponin have provided physicians with a new tool that, in combination with traditional techniques including ECG and clinical symptoms, significantly enhances their ability to accurately diagnose MI and triage patients.

As discussed here by experts at this summer's annual meeting of the American Association for Clinical Chemistry (AACC; Washington), there are about 6 million emergency department visits annually in the U.S. for chest pain, comprising about 6% of the total patient load in the ED. Four million are admitted to the hospital. Of those not admitted, 24,000 are subsequently found to have had an MI that was not diagnosed in the emergency room, according to Kristin Newby, MD, of Duke Clinical Research Institute (Durham, North Carolina), who discussed the topic at the AACC conference. About 830,000 of the admitted patients have a confirmed diagnosis of myocardial infarction, while 500,000 are subsequently diagnosed with angina. Some 910,000 of the admitted patients are diagnosed with other cardiac disorders (e.g., congestive heart failure), and 1.36 million are found to have non-cardiac conditions. As a result, there is a large demand for technologies that can allow physicians to rapidly risk-stratify chest pain patients and to do so as quickly as possible since those patients with a true MI will suffer increased heart damage the longer treatment is delayed.

While newer cardiac markers such as Troponin T and I have provided a major advance in diagnostic efficacy, there is still a need for improved markers that can provide an even earlier diagnosis, since troponin levels typically do not become elevated until five to six hours after the onset of an infarction. Myoglobin is often used along with troponin to provide an earlier indicator, but even that marker does not become elevated until four to five hours after onset, since, like troponin, it is released as a result of necrosis of cardiac tissue.

In addition, markers such as Troponin I or T and myoglobin are not specific for myocardial infarction or ischemic injury to heart tissue, but can also become elevated due to congestive heart failure, hypertension, shock, viral injury (myocarditis), trauma or exposure to toxins. A new development in the field is the introduction of markers of myocardial ischemia, which begin to be released before irreversible tissue necrosis occurs and can thus detect a developing MI within 30 minutes of the onset of reduced blood flow to the heart. In addition, a major market is emerging for markers such as BNP that can help determine if heart failure is involved in the patient's symptoms.

Perhaps most importantly from a public health perspective, significant progress is being made in the development of new indicators for cardiovascular disease risk, improving the ability of physicians to identify patients who are prone to have a heart attack and allowing preventative therapies such as statin drug treatment or diet and lifestyle modifications to be implemented. The most important development is the identification of high-sensitivity CRP as a new, independent risk assessment tool, which can be used in conjunction with lipid profiles to significantly enhance the ability of physicians to identify individuals who are at high risk for a heart attack. Other advances include increased availability of POC test systems for cardiovascular risk assessment, providing easier access to testing and expanding the proportion of the at-risk population that is screened.

Developments in the diagnosis of MI

A number of new products for improving the initial diagnosis of myocardial infarction have been introduced recently. Biosite's Cardiac ProfilER is a four-marker panel packaged in a single test cartridge that measures quantitative high-sensitivity Troponin I, CK-MB, myoglobin and BNP at the point of care. The $45 test is run on the Triage Cardiac MeterPlus, and was introduced during the second quarter of 2003. It provides capability not only for initial MI diagnosis, but also aids in risk stratification using results from the Troponin I and BNP measurements. Biosite has received FDA clearance for use of BNP in risk stratification of patients with acute coronary syndromes. In addition, BNP can be used in the diagnosis of congestive heart failure.

Another multi-analyte cardiac marker test has been developed for research use by Panomics (Redwood City, California) in collaboration with BioCheck (Burlingame, California). BioCheck is a supplier of ELISA immunoassay kits and antibodies for research, while Panomics is a developer of proteomic arrays. The two companies have developed the Transignal cardiac marker antibody array for the simultaneous quantitative measurement of hs-CRP, cardiac Troponin I, CK-MB, and myoglobin. Cost is $210 for a four-test kit. Future plans call for BioCheck to obtain FDA clearance for the product and introduce it to the clinical market.

A new qualitative cardiac marker testing product was exhibited at the AACC conference by SYNx Pharma (Toronto, Ontario) and LifeSign (Somerset, New Jersey). The product, called Nexus Dx MI by SYNx and LifeSign MI by LifeSign, also will be offered as a quantitative test in the future when used with an optional reader. SYNx is marketing the product in Europe, while LifeSign sells it in the U.S. Three versions of the test are available, including individual test cartridges for Troponin I, Troponin I/Myoglobin, and CK-MB/Myoglobin/Troponin I. Price per test (for a 20-test kit) is $21.95, $25.95 and $29.95 respectively. The test requires three drops of whole blood, plasma or serum and provides result in 15 minutes. The product is manufactured by Princeton BioMeditech (Monmouth Junction, New Jersey). According to LifeSign, the optional reader for producing quantitative cardiac marker results will be available in a few months. Its existing FDA-cleared test cartridges will be compatible with the new reader. A reader that can be used for quantitative measurement of various rapid tests, including cardiac markers, is available from Prima Biomedical (La Puente, California).

While the market for existing cardiac marker tests, including point-of-care and central lab tests for Troponin I, myoglobin, CK-MB, and BNP, is expected to continue to grow at about 15% per year over the next five years, physicians continue to search for additional tests that can help in the earlier diagnosis of acute coronary syndromes and that can aid in determining the most appropriate course of treatment. The recent introduction of troponin tests with increased sensitivity is one relatively recent advance that is enhancing the value of existing markers, but further improvements in diagnostic utility are expected to come from research aimed at identifying entirely new markers. In particular, tests that can detect myocardial ischemia are now available, and many more are under development, which promise to provide a definitive diagnosis of MI within 30 minutes of symptom onset.

Ischemia markers the next advance

At an AACC symposium on biochemical markers of ischemia, a number of next-generation tests were described that promise to provide earlier detection of a developing myocardial infarction. Table 2 describes the most promising cardiac ischemia markers. One new test already on the market in the U.S. detects Ischemia Modified Albumin (IMA), a compound that is formed when albumin comes into contact with ischemic tissue in the heart or another organ. IMA is detected with the Albumin Cobalt Binding (ACB) test developed and marketed by Ischemia Technologies (Denver, Colorado). The ACB test was cleared by the FDA in February, and launched in Europe about a year earlier. Recent studies have shown that the IMA test provides a more accurate risk stratification of ED chest pain patients than do existing markers coupled with physiological tests such as ECG monitoring. In particular, an elevated IMA result can indicate the need for continued serial monitoring of troponin levels and ECG stress testing in patients with a nondiagnostic ECG and negative troponin levels. Currently, such patients often are discharged if there are no other objective parameters that are indicative of a myocardial infarction. Use of the IMA test could thus help to prevent discharge of patients who are in need of treatment. IMA also provides a more definitive indication of those patients who can be discharged safely.

Robert Christensen, PhD, of the University of Maryland (Baltimore, Maryland), described a new marker, Nourin-1, that shows promise as a marker of cardiac ischemia and that may allow detection of a developing infarction before irreversible tissue damage occurs. The marker, under development by Nour Heart (Gaithersburg, Maryland), becomes elevated within five minutes after the onset of cardiac ischemia and acts to stimulate migration of cells such as neutrophils that are involved in tissue repair. The next step is development of an immunoassay for Nourin-1, allowing more extensive clinical assessment of the marker. Other new markers that have been studied for applications in earlier detection of MI include whole blood choline, glycogen phosphorylase BB, free fatty acids, pregnancy-associated plasma protein A (PAPP-A), fatty acid binding protein and platelet density. The latter test, described by Alan Wu, PhD, of Hartford Hospital (Hartford, Connecticut), uses the 2-D platelet analysis feature available on the Advia 120 hematology analyzer from Bayer Diagnostics (Tarrytown, New York). The test detects activation of platelets, which is believed to be a precursor to thrombosis that results in arterial blockage in MI.

Whole blood choline is another promising new early marker of myocardial infarction, described at the AACC conference by Oliver Danne, MD, of University Hospital Charite (Berlin, Germany). Whole blood choline appears to have a role in assessment of prognosis following MI, and also shows value when used in combination with troponin assays as well with high sensitivity CRP tests. The marker can complement troponin because it becomes elevated earlier, primarily because of its smaller molecular size, according to Danne. It also detects some patients with MI who are missed by troponin tests. When combined with hs-CRP, whole blood choline almost doubles the ability to predict adverse events accurately.

The continued expansion in the number of markers showing utility in MI diagnosis and risk stratification is analogous in at least one respect to the situation for in vitro tests for cancer. There, researchers are finding that large panels of markers, numbering in the hundreds in some cases, can provide more sensitive and specific detection of disease than existing single markers. In the case of MI diagnosis, the tests used by the clinical lab have expanded in recent years from CK-MB and, occasionally, various subtypes of that marker (CK isoenzymes), to include troponin and myoglobin, and most recently BNP. Experts at the AACC gathering speculated that much larger panels might be developed for MI diagnosis that could include inflammatory markers such as hs-CRP, markers of thrombosis, platelet activation markers, markers of ischemia, cardiac necrosis markers and perhaps markers of left ventricular dysfunction (e.g., BNP). Such panels could potentially be analyzed using emerging technologies such as proteomic microarrays, such as those developed by Ciphergen Biosystems (Fremont, California) or the microparticle-based flow immunoassay technology from Luminex (Austin, Texas), and provide a more comprehensive picture of patient status during MI than is possible with existing tests.

Success story in heart failure testing

Although BNP may have some applications in MI diagnosis and prognosis, its primary application is in the diagnosis of heart failure. The BNP test, as well as the related proBNP assay, is providing a quantum improvement in the ability of physicians to rapidly and accurately diagnose heart failure. Demand is particularly strong in the emergency department, where high patient loads, medico-legal liability concerns, hospital bed shortages and the complexity of diagnosing heart failure have led to high rates of misdiagnosis, delays in initiating treatment and poor patient outcomes. Patients with heart failure are often weak, making it difficult for them to comply with the requirements for a thorough clinical exam, and also may have co-existing conditions such as pneumonia that mimic the symptoms of heart failure. Biosite was the first to introduce a BNP test for clinical use, and the rapid rise in sales of the product, as shown in Table 1, provides strong evidence of the value of the test in improving patient management. Physicians discussing the use of the Biosite test at the AACC conference noted that new residents in emergency medicine find it difficult to imagine how heart failure patients were properly diagnosed in the past when the test was not available. Studies have demonstrated that the test provides a 30-fold increase in the odds ratio for heart failure diagnosis vs. all other clinical parameters customarily used to diagnose the disease.

Biosite has licensed rights to Beckman Coulter to make the BNP test available on laboratory immunoassay platforms, with plans to introduce a BNP assay on the Beckman immunoassay platform in 2004. In addition, Roche Diagnostics (Indianapolis, Indianapolis) introduced its NT-proBNP assay for the Elecsys analyzer late last year, and Bayer Diagnostics reported FDA clearance of a new BNP assay for its Centaur analyzer in June. Dade Behring (Deerfield, Illinois) acquired a non-exclusive license to the Roche proBNP technology in February. Although Biosite remains for now the only supplier of a point-of-care test for BNP, SYNx Pharma is developing a new POC test, the Nexus Dx CHF, using technology cross-licensed from Roche, that will be available as either a semi-quantitative test for rapid rule-out of congestive heart failure or, with a companion reader, as a quantitative POC test for both proBNP and Troponin I levels. The test will be priced between $22 and $25, and will be manufactured by Princeton BioMeditech. In addition to lower cost vs. the Biosite test, the SYNx Nexus Dx CHF test kit can be stored at room temperature, improving ease of use particularly in the emergency room.

Cardiovascular disease risk assessment

A third area of cardiovascular disease testing that is attracting widespread interest is new markers for risk assessment, to help identify those patients who are likely to have a heart attack, and to allow preventative measures to be taken at an early stage. Until recently, in vitro testing for cardiac disease risk was limited to measurement of blood lipid levels, including total cholesterol, LDL and HDL cholesterol, and triglycerides. Now, studies by Paul Ridker, MD, of Harvard Medical School and Brigham and Women's Hospital (both Boston, Massachusetts), have shown that high-sensitivity CRP provides significant additive information by identifying patients with normal lipid levels who nevertheless are at high risk for MI. CRP is produced in response to inflammatory processes, and its value in cardiovascular disease prognosis is related to the key role of inflammation of the blood vessel wall in plaque rupture. Because the CRP elevations associated with vascular inflammation are much smaller than those associated with infections or other inflammatory conditions, high-sensitivity assays must be employed, and patients may not have a pre-existing inflammatory condition that could bias the results.

There also is mounting evidence that CRP may play a causative role in cardiac disease, making it a potential target for therapeutic agents, but at present that is speculation. According to Ridker, however, many pharmaceutical companies are now developing anti-inflammatory agents that could be used for cardiovascular disease therapy. The potential at-risk population of individuals with elevated CRP levels is estimated to be about two-fold larger than that defined by cholesterol profiles, indicating a large target population for testing and for potential therapies.

As a result of the growing awareness of the value of CRP as a new marker of cardiovascular disease risk, a number of manufacturers have introduced high-sensitivity CRP assays, including Daiichi Pure Chemicals (Tokyo), Denka Seiken Co. Ltd. (Tokyo), Dade Behring, Diagnostic Products (Los Angeles, California), Mitsubishi Kagaku Iatron (Tokyo), Kamiya Biomedical (Seattle, Washington), Roche Diagnostics and Wako Diagnostics (Richmond, Virginia). Jokoh Co. Ltd. (Tokyo) is developing a new analyzer for POC measurement of hs-CRP that will be available at the end of 2003 pending FDA clearance. The Jokoh test will have a sensitivity of 0.5 mg/L and a turnaround time of five minutes, with features appropriate for use in a physician's office lab and similar settings. A number of other manufacturers are developing POC hs-CRP test systems for the European market. Based on a comparison of assay reproducibility and standardization discussed by Nader Rifai, PhD, of Boston Children's Hospital, at the AACC conference, the Iatron, Dade Behring, Wako and Diagnostic Products assays exhibited the best analytical performance in a study published in 2001. However, a new standard reference material has now been developed that is expected to allow all manufacturers to recalibrate their tests to provide a high level of inter-assay agreement.

New recommendations for the use of high-sensitivity CRP testing to assess cardiovascular disease risk were presented at the AACC conference by Gerald Cooper, MD, PhD, of the Centers for Disease Control and Prevention (Atlanta, Georgia). The recommendations do not call for screening of the general population with hsCRP, but instead advocate testing of those judged to be at intermediate risk as well as those with acute coronary syndromes or stable coronary disease. Implementation of hsCRP screening will drive additional growth in the market for cardiovascular risk testing products. Growth has been particularly strong in the point-of-care lipid testing segment, as shown in Table 3. POC lipid testing products include the LDX analyzer manufactured by Cholestech, the Three Minute monitor from Lifestream Technologies, the CardioChek and CardioChek PA from Polymer Technology Systems, home cholesterol tests from Home Access Health (Hoffman Estates, Illinois) and AccuTech (Vista, California), and the AccuTrend GC and AccuChek Instant Plus systems from Roche Diagnostics. Lipid tests such as total cholesterol, HDL and LDL cholesterol and triglycerides also are performed on a wide range of general-purpose chemistry analyzers in laboratory settings.

The total estimated worldwide market for products used in lipid testing is estimated at approximately $365 million for 2002. High-sensitivity CRP tests for POC testing systems such as the LDX are expected to become available by mid-2004, driving additional growth in the POC segment. The segment also will become more competitive, with new entrants such as Jokoh and Beijing Blue Cross Biotech (Beijing, China) entering the market in Asia and Innotrac Diagnostics (Turku, Finland) in Europe. Those suppliers all have plans to enter the U.S. market at a later date. High-sensitivity CRP testing is already helping to increase growth in the laboratory-based clinical chemistry market, a segment that has been essentially flat for the past few years.

Long term, continued expansion in the cardiovascular risk assessment testing market is anticipated, as the increased availability of POC testing systems makes screening more accessible for at-risk populations and as new risk markers continue to be developed. In addition to improved markers to help identify the approximately 50% of heart attack victims who have normal levels of lipids on screening tests, the market is likely to expand to include markers for the assessment of the risk of stroke. At least one potential new marker that could provide an early identification of patients who are prone to stroke is under development by CIS Biotech (Atlanta, Georgia). Biosite also is developing a panel of markers that can be used to help in the rapid diagnosis of stroke.