The rapid chromatographic immunoassay allows for accurate early detection of acute myocardial infarction.
Cardiovascular diseases are responsible for an estimated 17 million adult deaths in Western societies every year. Acute myocardial infarction (AMI), which causes almost 7 million deaths worldwide, is the most common and recurrent final expression of cardiovascular tissue damage.1
Since the 1950s, detection of cardiac proteins in plasma has been a means of assessing myocardial injury. In a patient sustaining ischemia, cells are irreversibly damaged and the plasma membrane ruptures. Intracellular proteins then leak out of the cell into the interstitial compartment, eventually appearing in vascular cavities. In 2000, the criteria for diagnosing AMI were redefined. The use of biochemical markers, especially more cardiac-specific proteins such as cardiac troponins T and I (cTnT and cTnI), was given increased importance.2
In order to enable early diagnosis and thereby improve AMI management substantially, a marker of myocardial damage should exhibit the following key characteristics:
• Rapid release from injured cells and subsequent appearance in plasma.
• Heart specificity and abundance in cardiac tissue.
• Low levels in plasma in normal, healthy controls.
One marker, human heart–type fatty acid–binding protein (H-FABP), has these properties. However, for this protein to be truly useful as an early cardiac marker to confirm or exclude an AMI diagnosis soon after the onset of symptoms, it requires a rapid assay system. Most immunochemical assays for H-FABP have limited utility for routine clinical practice, owing to their complicated procedures and need for skilled technicians. This article describes a novel credit card–style assay that may be suitable for the rapid detection of H-FABP.
Table I. Cardiac markers of myocardial infarction compared. (Click to enlarge.)
Cardiac markers now available are far from fulfilling important diagnostic requirements (see Table I).3-6 Creatine kinase MB isoenzyme (CK-MB) is relatively myocardial specific, but it lacks sensitivity because of its slow release. This rather large protein (86,000 amu) becomes elevated in the blood only 6–8 hours after the onset of typical symptoms. Myoglobin is released rapidly into the circulating blood, owing to its small size, and it is rapidly cleared by the kidneys, which makes it highly sensitive at early disease stages and good at detecting reinfarction. However, its abundance in skeletal as well as cardiac muscle makes it unspecific. The structurally bound proteins cTnT and cTnI, specific to cardiac muscle, first must be liberated from the myofibrillar matrix, and therefore often appear in the circulation only after a delay. Their concentration may take up to six hours to become elevated after an episode of myocardial damage, and may remain elevated for several days, which makes the detection of recurrent AMI episodes difficult.
Heart-type fatty acid–binding protein, a low-molecular-mass (15,000 amu) cytoplasmic protein abundant in heart muscle cells, offers great potential as a sensitive biomarker for early diagnosis of AMI.7-12 This relatively small protein is involved in cellular long-chain fatty acid metabolism.13 The concentration of H-FABP in the plasma of healthy persons is relatively low at 2–6 µg/L.14 It is released rapidly from damaged cells into the circulation and then cleared by the kidney; its plasma half-life is 20 minutes.7 H-FABP levels rise as soon as 1–3 hours after the onset of AMI, peak at 6–8 hours, and return to normal within 24–30 hours.
FABP is released from injured myocardium and eliminated from plasma in ways and at rates that are similar to those of myoglobin, but several clinical studies have revealed that H-FABP's performance is superior to that of myoglobin for early AMI detection and early estimation of infarct size.11,14–19 In a European multicenter study, H-FABP was found to be the most sensitive biochemical marker for early diagnosis of AMI.20 H-FABP is also superior to CK-MB and cardiac troponins in the early detection of ischemic myocardial necrosis.8,11,15,21,22 The fact that it shows a ratio of cytoplasmic to vascular concentration one order of magnitude higher than any other cardiac protein makes H-FABP the most sensitive and specific marker for AMI diagnosis within three hours from the onset of infarction.
The routine application of biochemical markers in AMI diagnostics will lead to a reduction in morbidity and mortality, and also to a reduction in hospital costs because patients with non-disease-specific symptoms can be sent home safely. A common drawback of the technology, however, has been delay in measurement, caused either by logistical complications or by labor-intensive analysis. Developments in point-of-care (POC) testing, though, do hold promise for enhancing patient care by making possible more-rapid test results.
POC tests for cardiac markers have been available for several years. Some have been qualitative, such as the Cardiac Status from Spectral Diagnostics Inc. (Toronto) and the Rapicheck for H-FABP from Dainippon Pharmaceutical Company, Ltd. (Osaka, Japan). Others have been semiquantitative, such as the Triage system from Biosite Inc. (San Diego). Quantitative tests include the Trop T from Roche Diagnostics (Mannheim, Germany); a troponin I electrochemical immunoassay offered by Abbott Point-of-Care (Abbott Park, IL); and the Ramp troponin I, CK-MB, and myoglobin system from Response Biomedical Corp. (Burnaby, BC, Canada).
A Rapid H-FABP Test
Figure 1. The CardioDetect FABP rapid test by rennesens GmbH (Berlin) is offered in the CardioDetect self version (a) for self-testing (its two separate test fields allow one repetition) and the CardioDetect med version (b) for hospital use (containing one test field and a label on which to note patient data). The test is also now being manufactured in China by Shenzhen Kang Sheng Bao Bio-Technology Co., Ltd. (Shenzhen).
The focus of the study reported here is a one-step FABP immunotest called CardioDetect that was developed through a collaboration between 8sens.biognostic AG and rennesens GmbH (both of Berlin) (Figure 1).23–25 This commercially available rapid test, which carries CE mark approval, is a chromatographic immunoassay designed for quantitative determination of H-FABP in whole blood, plasma, and serum samples. Following introduction of the sample onto the test strip, the result is available within 15 minutes. The whole blood immunotest requires no sample pretreatment; thus, it can be used in emergency situations. When combined with testing for the well-established markers troponins, this FABP assay may allow more-accurate targeting of appropriate therapy than the current diagnostic tests for troponins, myoglobin, and CK-MB, and electrocardiography, and at considerable cost savings. It is stable and can be stored at 4°–8°C for up to one year without any loss of activity.
The test is available in two credit card styles. CardioDetect med is designed for use by medical doctors, and CardioDetect self is for self-testing.
Assay Development. In developing the assay, researchers produced recombinant FABP, used as standard in the calibration curves, in-house.26 To obtain sufficient amounts of protein for in vitro studies, human H-FABP was expressed in Escherichia coli and purified in one step using anion-exchange chromatography. The biochemical properties of the recombinant protein were then compared with the protein isolated from human heart.27
Monoclonal anti-H-FABP capture and detector antibodies also were produced in-house. The antibodies were purified from ascites by ammonium sulfate precipitation (40% on a volume-per-volume basis) and by affinity chromatography using a protein G-Sepharose column (Amersham Phar- macia Biotech AB; Uppsala, Sweden).
Goat antimouse IgG was purchased from Arista Biologicals Inc. (Allentown, PA), which also prepared detector antibody conjugated to colloidal gold. Detector antibody conjugated to horseradish peroxidase was prepared in-house with a peroxidase-labeling kit from Roche Diagnostics Corp. (Indianapolis).
Test Principle. In the developed assay, the reaction is initiated by adding three drops of venous or finger blood (equivalent to 100–120 µl) to the well of the test device. Either heparin or citrate may be used as an anticoagulant. FABP in plasma binds to the colloidal gold–labeled antibody (optical density value at 540 nm = 12; 5.0 µl per strip), and the resulting complex overflows the nitrocellulose membrane where the capture antibody (2.0 µg per strip) is immobilized in a capture zone. Finally, a sandwich complex consisting of colloidal gold–labeled antibody, H-FABP, and capture antibody is generated.
The concentrated gold particles appear as a red band. The intensity of the color and the speed at which it develops depend on the concentration of H-FABP in the blood. Unreacted colloidal gold– labeled antibody may thereafter combine with goat antimouse IgG (1.5 µg per strip) immobilized distally to the capture band. The formation of this control band indicates the unimpeded flow of plasma through the device.
Clinical Relevance and Indications
Figure 2. The CMOS-based Cardioquant test reader by sitec GmbH (Brandenburg, Germany).
Users of CardioDetect can gauge the test-strip results visually with the naked eye. The results can also be quantified by means of a complementary metal-oxide semiconductor (CMOS)–based test reader called the Cardioquant by sitec GmbH (Brandenburg, Germany) (see Figure 2). This device uses a white light–emitting diode as the light source and can be configured for handheld or computer-controlled operation.
Figure 3. Test results are rated as positive when H-FABP bands of varying degrees of red appear. When only a control band is observed, the result is considered negative. If no control band appears, the test should be discarded.
If the test result is positive, two red lines will be visible, one at the area of the strip labeled Control and one at Result (or h-FABP on the CardioDetect med card) (see Figure 3). This indicates that the concentration of H-FABP in the sample is above the threshold value of 7 µg/L and that an AMI has occurred. If the result is negative, then only one red line will be visible at Control. This reading indicates that the concentration of H-FABP in the sample is equal to or below the threshold value and that an AMI has not occurred. (However, if pain continues, the patient should, of course, see a doctor for further examination.) A test is invalid and should be discarded if no lines are visible or if a line only at Result (h-FABP with CardioDetect med) is visible. In such a case, the patient should repeat the test immediately and make sure that enough blood is applied.
The reading zones at the bottom of Figure 3 show, from left to right, the results for a non-AMI patient (–) and AMI patients with H-FABP levels of 20 and 40 µg/L (+), 80 µg/L (++), and 160 µg/L (+++). The intensity of the H-FABP band apparently is directly related to the H-FABP concentration in plasma.
Investigators compared the analytical performance of CardioDetect with that of a modified one-step sandwich enzyme-linked immunosorbent assay (ELISA) using 434 plasma or serum samples taken from 218 patients admitted to the Coronary Care Unit and Chest Pain Unit of the Prince of Wales Hospital in Hong Kong presenting with chest pain and suspected AMI. The samples covered an H-FABP concentration range of less than 2 µg/L through approximately 3000 µg/L. The detection limit of the ELISA was 0.2 µg/L. This yielded a coefficient of variation of less than 10% in inter- and intraassay verifications. From each sample, 80 µl was applied to the test field of a CardioDetect self card assay and analyzed by inspection at 15 minutes after application. Whenever bands appeared at both the Result and Control zones, the result was rated as positive, with a deep-red band being very positive (+++), a medium-red band being moderately positive (++), and a slightly red band only technically positive (+), as shown in Figure 3. When only the Control band registered, the result was rated as negative. When no Control band was observed, the test was discarded. The investigator was blinded with regard to the H-FABP concentrations applied and the labeling code used.
Comparison with ELISA. Of the 434 samples tested by ELISA, 194 had H-FABP concentrations no greater than 7 µg/L (the threshold level); 54 samples had concentration above threshold but no greater than 50 µg/L; 50 contained H-FABP at above 50 µg/L through 100 µg/L; and 136 had concentrations above 100 µg/L. Of the corresponding samples tested with the CardioDetect self, 63% with H-FABP concentrations of >7 to 50 µg/L, 78% in the range of >50 to 100 µg/L, and 94% containing H-FABP at above 100 µg/L yielded rapid test results in agreement with the ELISA results.
Figure 4. The CardioDetect self and the FABP ELISA were used to test 434 samples of plasma or serum from 218 suspected AMI patients. The illustration charts the CardioDetect results that fell within and outside of the ranges of H-FABP concentration associated with readings deemed negative (<_ 7 µg/L), just positive (>7 to 50 µg/L), more positive (>50 to 100 µg/L), and highly positive (>100 µg/L).
In tests performed using CardioDetect self, 4.1% of samples (18 of 434) with H-FABP concentrations above 7 µg/L showed negative results—that is, no band at the Result zone (see Figure 4). All of these false-negative samples contained H-FABP concentrations below 20 µg/L. There was no sample with H-FABP at 7 µg/L or less showing a positive result.
Precision at the Detection Limit. The precision of the CardioDetect at the detection limit was tested via five repetitive measurements of blood samples with H-FABP concentrations of 5, 7, 10, 15, 20, 40, and 80 µg/L analyzed at 5, 10, and 15 minutes (Figure 5). The speed of development of an H-FABP–positive band depends on the H-FABP concentration in the sample. When H-FABP levels are 80 µg/L or higher, a band develops in all cases within 5 minutes. At 15 minutes, the results were negative in all patients with H-FABP concentrations of 7 µg/L or less and positive in all patients with H-FABP concentrations of 15 µg/L or more.
Figure 5. Precision of the CardioDetect H-FABP immunoassay at the detection limit,
with an indication of the speed of color development, as determined by five repetitive
measurements of each of seven blood samples taken at 5-minute intervals.
In the range of 7–20 µg/L, subjective assessment of a band's positive quality varied. Positive results were declared more frequently as H-FABP concentration increased. When 10 different people assessed the same test strip, they found similar results. All agreed that the result was positive whenever H-FABP concentrations were at least 15 µg/L and all saw a negative result anytime the concentration was no higher than 7 µg/L.
Table II. Analytical performance consistency of the one-step rapid immunoassay. Ten test cards were used for each of the four analyses. (Click to enlarge.)
Assay-to-Assay Consistency. Blood samples with H-FABP concentrations of 10, 25, 100, and 500 µg/L were analyzed to assess intraassay precision. For this purpose, investigators tested each sample with 10 test cards from the same CardioDetect production batch (either CardioDetect med or CardioDetect self). Interassay precision was assessed through measurement of each sample with 10 test cards taken from three different batches of CardioDetect med and from two different batches of CardioDetect self. The intraassay coefficient of variation (CV) was 6.1–16.8%, and the interassay CV was 8.0–16.5% (see Table II).
The Hong Kong hospital study was approved by the Clinical Research Ethical Committee of the Faculty of Medicine at the Chinese University of Hong Kong. All patients gave written informed consent to participate after receiving a thorough explanation of the study protocol.
Of 218 patients presenting with chest pain and suspected AMI, 94 patients (75 males and 19 females) with an average age of 63.1 years (±11.9 years) were diagnosed with AMI. The other 124 patients (80 males and 44 females), aged 62.1 years (±12.1 years) on average, were diagnosed as not having experienced AMI.
Of those 218 patients, 190 (87.2%) were admitted to the hospital within six hours of the onset of symptoms. Of these, 132 presented within three hours following the onset of chest pain. (More cardiac patients in Hong Kong are admitted to hospitals after only a short delay than is the case in Europe and the United States.28,29)
Diagnostic Reliability with Plasma and Serum Samples. The analytical performance of CardioDetect self as correlated with the conventional ELISA test method was discussed in the preceding section. Using the same set of plasma or serum samples from the Hong Kong patients, the investigators evaluated the diagnostic performance of the CardioDetect self with reference to the time interval between the onset of symptoms and analysis.
Table III. Clinical comparison of the area under the ROC curve for various diagnostic tests used for detection of myocardial infarction. (Click to enlarge.)
The areas under the receiver operating characteristic (ROC) curves for the rapid test, the FABP ELISA, the cardiac troponin I ELISA, and the CK test were compared (see Table III). (In signal detection theory, these curves are a graphical plot of sensitivity versus specificity for a binary classifier system as its discrimination threshold is varied.) The measurement performance of the one-step immunoassay was observed to be similar to that of the FABP ELISA. The areas under the ROC curves for the CardioDetect and the FABP ELISA were significantly greater than those for cTnI and CK within six hours of the onset of symptoms, meaning that FABP is a cardiac marker that provides a better diagnostic classification of AMI likelihood than cTnI and CK in the early phase.
Diagnostic Reliability with Whole Blood Samples. In addition to assessing the clinical performance of CardioDetect self in Hong Kong, investigators tested patients suspected of having AMI in the emergency department or corresponding clinical department of two specialized European hospitals, this time using CardioDetect med. They studied blood samples obtained from 38 patients (21 AMI and 17 non-AMI) admitted to the hospital in Bernau, Germany, near Berlin, with chest pain. Using an upper reference level of 7 µg/L, they found the specificity of the test to be 94%. Both sensitivity and negative predictive value (NPV) were 100%; that is, 100% of non-AMI patients can be excluded, with no false-negative results. Also, 171 patients (97 AMI and 74 non-AMI) at the German Diabetes Research Institute in Düsseldorf were investigated. Sensitivity and NPV were again 100%, and the evaluation this time showed specificity to be 85.1%.
The rapid H-FABP assay has been tested in Chile, France, Malaysia, Poland, Spain, and the Czech Republic, as well, exhibiting similar encouraging diagnostic performance in those studies.
Limitations. H-FABP may be elevated in patients with renal deficiency owing to the impaired performance of their kidneys. This may lead to a false-positive test result. Also, in patients with angina pectoris, a false-positive result cannot be excluded.
A small amount of H-FABP is present in skeletal muscle. Therefore, false-positive results are technically possible for athletes and for individuals who have exercised vigorously prior to the testing.
Rapid bedside immunotests involving specific monoclonal antibodies will allow H-FABP to be used in clinical settings, together with other clinical tools such as troponin assays, to diagnose and treat cardiac patients as soon as possible so that mortality and morbidity can be decreased.
At present, attention is focused on defining those cardiac proteins, or combinations of proteins, that exhibit a high sensitivity and specificity for AMI detection, especially in the first few hours after patient admission. In order to minimize the risk of falsely excluding patients who are actually experiencing ongoing AMI, the investigators whose work is reported here are now collaborating closely with an accredited Asian company to develop a system for combined measurement of two biochemical markers, H-FABP and cTnI, that may provide the optimum diagnostic performance. At the same time, they continue to investigate the performance of H-FABP, via the CardioDetect assay, for early diagnosis of AMI in patients presenting with acute coronary syndromes in emergency departments, as well as for excluding non-AMI cases.
Further, a simple, semiquantitative rapid test for simultaneously detecting heart attack through H-FABP and predicting cardiovascular disease risk by means of a C-reactive protein assay, involving simply counting the number of red lines on a strip, is now under development. This could enable doctors to predict the extent of a patient's risk early on and then prescribe suitable heartattack prevention therapy.
The potential of this new rapid and sensitive H-FABP immunotest in the detection of myocardial infarction is considerable. It may help physicians to diagnose AMI more accurately and earlier, leading to improved treatment that will reduce patient morbidity and mortality and prevent unnecessary hospital stays for non-AMI patients. The associated economic and human advantages would be important. Its simplicity could even make it a user-friendly test for application by chronic patients at home. This sophisticated and effective diagnostic tool could enable both physicians and patients to control diseases that now cause extensive disability and loss of life worldwide.
|Clockwise from top left : Cangel P. Y. Chan, PhD, is a research director at R&C Biogenius Ltd. (Hong Kong). Matthias Lehmann, PhD, is director of R&D, and Ilka Renneberg is managing director at 8sens.biognostic AG (Berlin). Jürgen Ziegler is chief director of rennesens GmbH (Berlin). George W. H. Cautherley is managing director of R&C Biogenius Ltd. (Hong Kong). Jan F. C. Glatz, PhD, is a professor in the department of molecular genetics at the Cardiovascular Research Institute Maastricht at Maastricht University (Maastricht, The Netherlands). Reinhard Renneberg, PhD, is a professor in the biosensors and bioelectronics laboratory at the Hong Kong University of Science and Technology department of chemistry. The authors can be reached at firstname.lastname@example.org, email@example.com, firstname.lastname@example.org, email@example.com, firstname.lastname@example.org, email@example.com, and firstname.lastname@example.org, respectively.|
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