Cardiac markers and point-of-care testing: the perfect fit
Each year, more than six million Americans visit clinics and emergency departments reporting chest discomfort. These patients must be evaluated accurately, quickly, and efficiently to optimise outcomes, conserve healthcare resources and minimise the likelihood of litigation for improper diagnosis. It has been reported that each year up to 70 per cent of patients admitted to coronary care units for suspected acute myocardial infarction (AMI) are later discharged with a different diagnosis costing billions of dollars.

Biochemical markers are of increasing importance in diag-nostic strategies for ruling in and ruling out AMI, particularly when electrocardio-graphic(ECG) findings do not allow a diagnosis. During the 1990s, physicians showed great interest in the utilisation of biomarkers to facilitate the diagnosis and treatment of patients with acute coronary syndromes. Today, cardiac markers are a multimillion-dollar industry. In the Western world, serial testing for cardiac markers are performed on nearly every patient presenting with chest pain.

Biochemical markers are of increasing importance in diagnostic strategies for ruling in and ruling out acute myocardial infarction (AMI), particularly when electrocar-diographic (ECG) findings do not allow a diagnosis. Point-of-care (POC) or ‘near-patient’ testing allows for diagnostic assays to be performed at the site of patient care delivery. The biochemical markers that are commonly used by physicians to aid in the diagnosis of AMI are myoglobin, CK-MB, troponin I, and troponin T. Currently available POC assays possess comparable diagnostic performance to laboratory based cardiac marker assays providing the opportunity for POC testing to evolve into the standard-of-care for evaluating more than six million American patients with chest pain. Full acceptance of this relatively new technology will not be realised until users reach a comfort level where utilisation of these devices is foolproof and they have faith in the results.

Point-of-care (POC) or ‘near-patient’ testing allows for diagnostic assays to be performed at the site of patient care delivery such as the ED, chest pain evaluation center, or intensive care unit (I C U ). Compared with centralised laboratory testing, POC testing provides for rapid clinical decision making by reducing the time spent ordering tests, collecting and transporting samples, as well as retrieving data. Increasingly, POC tests for cardiac markers are being researched, developed, and put into clinical use. Most of these new POC devices have common attributes in that they use small quantities of whole blood; return results in 10 to 30 minutes; and demonstrate accuracy and precision comparable to laboratory based analysers.

Acute Myocardial Infarction and Cardiac Markers Typically, AMI is the result of a sudden occlusion that decreases blood flow to a portion of the myocardium, causing cell death. In the majority of cases, the mechanism accountable for the occlusion is a focal rupture of the surface of an atherosclerotic plaque causing a thrombus to form at the point of rupture.

The World Health Organisation’s (WHO) criteria for diagnosing AMI was developed in 1979 and specifies that two of the following three criteria must be met:
– characteristic chest pain
– diagnostic ECG changes
– elevation of serial enzyme changes.

Many of the biochemical markers (described below) that came into use after the publishing of this criteria are not themselves enzymes, such as myoglobin and the troponins. This has lead to a call for revision of the criteria.

Serial measurement of biochemical markers is now universally accepted as an important determinant in AMI diagnosis. In addition, new biochemical markers have demonstrated significant benefit in the risk stratification of patients presenting with unstable angina, as well as in outcome prediction and therapy selection.

The Markers

The ideal cardiac marker is one that has high clinical sensitivity and specificity, appears early after AMI, remains abnormal for several days after AMI, and can be assayed with a rapid turnaround time. Today, there is no single marker capable of meeting all these criteria necessitating a multi-analyte approach. The biochemical markers that are commonly used by physicians to aid in the diagnosis of AMI are myoglobin, CK-MB, troponin I, and troponin T. As is depicted in the graph below, cardiac markers follow a specific, predictable pattern of release kinetics following a coronary event. The differences in the time that it takes each marker to reach peak concentration has made it standard practice for clinicians to make use of at least two different markers in tandem; an early marker, and a later one.

Myoglobin: Myoglobin is a 17.8 kDa oxygen binding heme protein present in both cardiac and skeletal muscle. Cell injury during AMI releases myoglobin into the blood circulation. As a small molecule, myoglobin diffuses quickly into the circulatory system and can thus be detected soon after an AMI, reaching twice unaffected serum concentrations within two to three hours after AMI and peaking within four to six hours. Quick release into the blood stream makes myoglobin a valuable, early screening test for AMI. However, as it is not cardiac specific, elevated levels should be interpreted with caution if the patient being evaluated has renal dysfunction or skeletal muscle injury.

Creatine Kinase and CK-MB:
Creatine kinase is an important enzyme regulator of high energy phosphate production and utilisation within contractile tissues. CK has different isoforms (isoenzymes) depending on the tissue from which it originates: CK-BB, the brain component; CK-MM, the skeletal muscle compo-nent; and CK-MB, produced primarily in the myocardium. Total CK, being made up of these three molecular forms originating from a variety of tissues, is a poor indicator of AMI. However, CK-MB (86 kDa), is more specific to coronary events and has been the gold standard among biochemical markers for diag-nosis of AMI. Serially measuring CK-MB is an established criteria for myocardial cell injury when ECG is inconclusive with levels reaching twice the unaffected concentrations within five to six hours after the onset of chest pain and peaking in 12 to 24 hours. As with myoglobin, there are a number of non-AMI related pathologies (e.g. urinary tract infection, seizures, congestive heart failure) that may result in increased levels of CK-MB, hence caution must be used in the interpretation of results.

The Troponins: In striated muscle, the thin filament contains a calcium regulating protein complex containing three polypeptides, troponin C, I, and T (all < 30 kDa). Only TnT and TnI are sensitive and specific for cardiac damage.

Troponin T, which has a muscular tissue component, may elevate in muscle injury or renal failure whereas troponin I offers greater specificity for AMI assessment because it is exclusive to heart muscle tissue. TnI kinetics following AMI are similar to CK-MB in that it takes four to eight hours to increase above the upper reference limit, peaks between 14 and 36 hours and may remain elevated three to seven days after the event. TnT mimics the early release kinetics of TnI, but may remain elevated for as long as three weeks. Late clearance of troponin make it a good marker for diagnosing AMI at later stages but reduce its utility in assessing recurrent AMIs.

In 1999, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) released evidence and suggestions on the use of biochemical markers for the triage diag-nosis of acute coronary syndromes. For hospitals with a protocol for rapid AMI ruling out, in the absence of definitive ECG evidence, there is a sampling frequency recommended for the detection of AMI by biochemical markers.

Using upper reference limits as a diagnostic threshold, the negative predictive value for myoglobin is approximately 100% four hours after admis-sion allowing for early exclu-sion of AMI. Positive results for troponin lead to triage of a patient to the appropriate level of care, without the need for necessarily completing the sequence of blood samples.

Point of Care Technology

Antibodies have been in use as diagnostic reagents for over 50 years, but the modern day immunoassay, now one of the fastest growing technologies in clinical diagnostics, owes itself to the work of Yallow and Berson.

Point of care immunoassay devices can be classified as either qualitative or quantita-tive. Qualitative POC devices, such as home-pregnancy tests, only yield a positive/negative result depending on whether the analyte of interest meets or exceeds a predefined threshold. If the test is positive, the result is visibly recognised, usually as a change in colour. Quantitative POC devices analyse the specimen and provide a number correlating to the amount of analyte in the sample. The choice of which type of assay to use depends upon the needs of the user.

Qualitative: Spectral Diagnostics’ (, Toronto, Canada) Cardiac STATus combination test employs solid-phase chromatographic immunoassay technology to simultaneously detect the presence of Troponin I, CK-MB, and myoglobin above an established cutoff level in human blood, serum and plasma samples. After a specimen has been dispensed into the sample well, plasma or serum is transferred into a region containing monoclonal antibody-dye conjugates and biotinylated polyclonal anti-bodies. These antibodies bind to their respective analytes in the sample to form complexes, which migrate through the reaction strip. The antigen/antibody dye complexes are then captured by immobilised streptavidin in the capture area. Additional protein dye conjugates not bound in the test area are later captured in the control area. Visible purplish horizontal bands will appear in the test and control areas if the level of the analytes is above the established cutoff.

The Cardiac STATus combination assay provides a qualitative analytical test result. The qualitative nature of this assay does not provide information about change – either the rise or fall – in the concentration of the analytes with single testing.

Fixed Quantitative: The Dade Behring (www.dade-behring. com, Deerfield, IL) Stratus CS, a fluorometric enzyme immunoassay analyser based on solid-phase radial partition immunoassay technology, performs quantitative measurements of CK-MB, myoglobin, and troponin I in whole blood samples collected using lithium heparinate as anticoagulant. The samples are processed automatically by the analyser, which provides the option of centrifugation if heparinized plasma is not dispensed directly into the sample cup for analysis. In this procedure, dendrimer linked monoclonal antibody is added to the centre portion of a square piece of glass fibre paper in the TestPak. This antibody recognises a distinct antigenic site on the cardiac marker of interest. Sample is then added onto the paper where it reacts with the immobilized anti-body. After a short incubation, a conjugate consisting of enzyme labeled monoclonal antibody directed against a second distinct antigenic site on the cardiac marker is pipetted onto the reaction zone of the paper. During this second incubation period, enzyme-labeled antibody reacts with the bound cardiac marker forming an antibody-antigen labeled antibody sandwich. The unbound labeled antibody is later eluted from the field of view of the analyser by applying a substrate wash solution to the centre of the reaction zone. The enzymatic rate of the bound fraction increases directly with the concentra-tion of analyte in the sample. The reaction rate can then be measured by an optical system that monitors the reaction rate via front surface fluorescence. All data analysis functions are performed by the micro-processor within the analyser.

The Stratus CS is designed for emergency departments and other locations where quick and effective diagnosis is required. It is also suited for low volume cardiac testing performed in a clinical laboratory.

Portable Quantitative: The RAMP System by Response Biomedical (, Vancouver, Canada) consists of the following components: (i) a portable fluorescence reader that is used to quantify antibody antigen complexes, and (ii) a disposable cartridge that houses an analyte-specific immunochromato-graphic strip.

The RAMP assay cartridge contains a mylar-backed nitrocellulose strip on which the assay runs. On the membrane strip are two populations of latex particles each in dependently fluorescently labeled and tagged with specific monoclonal anti-bodies to generate two independent signals during the assay reaction. One of these is the test reaction and the other is an independent internal standard. An internal standard has been incorporated into the RAMP tests that run concurrently with every test assay to compensate for test-to-test variations in membrane and sample properties.

The RAMP Reader incorporates two detectors, one for each wavelength of dye incorporated in the latex particles. When the reaction in the cartridge is complete, the reader scans the test strip, causing the dyes to fluoresce at two independent wavelengths that can be detected by the reader. The RAMP reader determines the absolute concentration of analyte. To perform this task, the reader calculates the ratio between the concentrations of latex particles in the test and internal standard reactions, refers to an analyte-specific calibration curve, and converts this ratio to analyte concentration.

To perform a test, the operator places an anticoagulated whole blood sample into the well of a test cartridge and inserts the cartridge into the reader. After application, the sample is drawn by capillary action along the membrane through the contact and internal standard zones. The analyte present in the sample will interact and bind with the antibodies at the contact zone. The reader’s two detectors separately measure the amount of fluorescence emitted by the complexes bound on the detection and internal standard zones. To establish a quantitative reading, the reader calculates the ratio between the two readings. By calculating the final test result as a ratio between the two measurements, the RAMP system automatically accounts for variations in sample and membrane properties. A blank or abnormal reading from the internal standard automatically invalidates the test, acting as an automatic quality control.

Within 10 minutes from cartridge insertion into the reader the assay is complete, and the test result appears on the LCD readout. The result can also be stored, printed, or uploaded to a laboratory or hospital information system.

The RAMP System is being developed for use in all POC settings, and due to its compact size, and battery operation, it is a truly portable testing system. The RAMP system is proceeding to clinical trials for FDA submission in 2001. The system is designed to accommodate a broad spectrum of analytes, of which myoglobin will be the first to go to trials. Tests for CK-MB and Troponin I will follow soon after.


The ultimate goal of POC testing is to improve patient outcomes. Full acceptance of this new technology will not be realised until a comfort level is reached by users where utilisation is fool-proof and they have faith in the results.

New POC assays possess comparable diagnostic performance to laboratory based cardiac marker assays providing the opportunity for POC testing to evolve into the standard of care for evaluating over six million American patients presenting with chest discomfort.

The decision as to which device to use, which markers to employ and when to perform the assays must be made by each individual institution.

Recommendations by societies such as the IFCC will assist in the decision making process but there are no clearly obvious answers. New biochemical markers will continue to be developed as new information is acquired on the pathophysiology of AMI and as new therapeutic strategies develop.

>> Supplied by Joanne Stephenson & Paul Harris, PhD, Response Biomedical Corporation, British Columbia, Canada. For more details phone (604) 681-4101.


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