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Articles |
1
University of Lübeck, Medizinische Klinik II, 23528 Lübeck, Germany.
2
University of Heidelberg, Innere Medizin III,
D-69115 Heidelberg, Germany.
3
Boehringer Mannheim, D-68298 Mannheim, Germany
a Address correspondence to this author at: Medizinische Universität zu Lübeck, Medizinische Klinik II, Ratzeburger Allee 160, 23538 Lübeck, Germany. Fax 49-451-5006437.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods and Results: The cTnT test strip reader measures, via a CCD camera, the reflectance of the signal line. For quantitative analysis, a calibration curve was constructed from 1030 samples of 252 consecutive patients with acute coronary syndromes. In a method comparison of 140 samples, the quantitative results of the cTnT test strip reader correlated closely with the results of the cTnT ELISA (r = 0.98; y = 0.85x + 0.002). Within-run and day-to-day (n = 10) mean CVs were between 11% and 16%, respectively. The cross-reactivity with skeletal troponin T was <0.02%. In patients with myocardial infarction, 45% and 91% of all samples were positive on admission and at 4–8 h after the onset of symptoms, respectively. ROC curve analysis demonstrated a comparable efficiency of the cTnT test strip reader and the laboratory-based cTnT ELISA in patients with suspected myocardial infarction.
Conclusions: It is now possible to quantitatively determine cTnT at the patient's bedside with a rapid and convenient test device. This will facilitate the diagnostic work up of patients with suspected myocardial cell necrosis.© 1999 American Association for Clinical Chemistry
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The visual assessment of the available whole blood assay allows a yes or no statement without definite information regarding the concentration of cTnT in blood. Although this qualitative assay is sufficient for clinical decisionmaking processes, some prognostic information inherent to the concentrations of circulating cTnT is lost. Furthermore, interindividual variability of visual assessment of the test strip at the detection limit of the assay may cause substantial analytical error (2). We now present an improved bedside assay for cTnT and a newly developed reader to overcome the limitations of previous tests and to enable reliable quantitative and rapid testing for cTnT. This diagnostic tool will facilitate the triage and therapeutic decisionmaking process in the treatment of chest pain patients.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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quantitative test strip reader
The measuring unit of the quantitative test strip reader (Cardiac
reader; Boehringer Mannheim) contains a CCD camera that
optically records the detection zone of the immunochemical test strip.
The signal and control lines in the detection zone are recognized by an
evaluation algorithm. The intensity of the signal line, determined by
measuring its reflectance, is directly proportional to the
concentration of cTnT. A negative test result (absence of a signal
line) produces a reflectance of 100%. The test requires a sample
volume of 150 µL of heparin-treated venous blood and has a reaction
time of 12 min.
calibration curve
To establish a calibration curve for the cardiac reader, we
measured 1030 samples by ELISA and by a functional model of the cardiac
reader, which worked on the basis of the reflectance only. The samples
were obtained from 252 patients with chest pain and suspected
myocardial infarction (for details, see the section on clinical
analysis below). The cTnT concentrations measured by ELISA were 0–71
µg/L (median, 0.02 µg/L; 25th percentile, 0.0 µg/L; 75th
percentile, 0.66 µg/L).
The calibration between the percentage of remission measured with the test strip reader and the cTnT concentration measured with the comparison method ELISA cTnT curve revealed a nonlinear relationship. The measured reflectance was converted via the calibration curve into a cTnT concentration.
analytical method comparison
The analytical performance of the cardiac reader was compared with
the standard ELISA by the use of 140 heparin-treated blood and serum
samples from 140 patients with acute coronary syndromes.
imprecision experiments
The within-run imprecision was determined by 10-fold measurements
with lyophilized control materials and with different blood samples
supplemented with cTnT. The day-to-day imprecision was obtained
from 18 repetitive measurements on 18 subsequent days with controls.
cross-reactivity testing
Blood samples were supplemented with skeletal troponin T up to a
concentration of 500 µg/L in combination with three different cTnT
concentrations (0, 0.2, and 1 µg/L) [preparation as described in
Katus et al. (5)].
clinical analysis
The clinical study group comprised 64 healthy volunteers and 252
consecutive patients with suspected acute coronary syndromes seen at
the emergency room. The clinical diagnoses in the patients, based on
WHO criteria (6)(7), with chest pain were as
follows: (a) acute myocardial infarction (AMI; 37 patients,
351 samples), (b) unstable angina pectoris (44 patients, 354
samples), or (c) no evidence of acute myocardial ischemia
(171 patients, 325 samples).
All patients gave informed consent to participate in the study after thorough explanation of the study protocol. This investigation was approved by the ethics committee of the University of Heidelberg.
blood sampling
From each patient, heparin-treated blood and serum were collected
on admission; 3, 6, 12, and 24 h after admission; and once per day
until day 10 or until discharge. All bedside assay measurements and
creatine kinase (CK) activity determinations were performed immediately
after a blood specimen was obtained. cTnT ELISA and CK-MB mass
measurements were performed from frozen serum within 2 weeks after the
samples were collected.
determination of cardiac marker molecules
The cTnT rapid assay (Boehringer Mannheim) was measured
quantitatively on the test strip reader (Boehringer Mannheim). Each
test strip was rechecked visually.
The cTnT ELISA (Enzymun Troponin T-Test; Boehringer Mannheim) was performed according to the manufacturer's instruction for use (4). The cutoff value used for the cTnT ELISA was 0.1 µg/L.
Total CK activity was determined with a ClinChem Analyzer (Miles) and the reagents provided by the manufacturer. The upper reference limit for total CK activity is 80 U/L in men and 75 U/L in women (25 °C).
CK-MB mass concentrations were determined using the Creatine Kinase-MB Fluorometric Enzyme Immunoassay adapted to the Stratus analyzers (Baxter Diagnostics). The upper limit of normal used for CK-MB mass determinations is 4.8 µg/L. As a cutoff value for clinical evaluations, we used 9.6 µg/L, which is twice the upper limit of normal.
statistics
ROC curve analyses were performed using ASTUTE software (DDU
Software).
The relationship between the percentage of remission values of the rapid assay and the concentrations of cTnT ELISA was described using nonlinear regression analysis. The regression function was established using Easy software (Boehringer Mannheim).
Concentration values are given as means and SD or as medians and ranges (25th and 75th percentile).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The data pairs were used for nonlinear regression analysis to
obtain a calibration curve for the rapid assay, which is shown in Fig. 1
. The cutoff concentration of 0.1 µg/L for the cTnT ELISA
corresponds to a remission value of 97% in this calibration model.
This was also confirmed by ROC curve analysis.
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Imprecision.
The within-run imprecision experiments (n =
10) with the cTnT rapid assay yielded CVs between 9% and 14% for cTnT
concentrations between 0.16 and 1.24 µg/L. The CV for day-to-day
imprecision, performed as 18 repetitive measurements on 18 subsequent
days, was between 15% and 18% (Table 1
).
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Correlation and accuracy in method comparison.
Compared with
the reference method, cTnT ELISA, the results obtained with the cTnT
rapid assay show a high correlation at concentrations of 0.1–3 µg/L
(r = 0.98). The accuracy of the test was ± 15%
compared with the cTnT ELISA (y = 0.85x +
0.002; Fig. 2
).
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Cross-reactivity with skeletal troponin T.
The addition of up
to 500 µg/L of troponin T purified from human skeletal muscle to
cTnT-free samples did not lead to a detectable signal line on the test
strip. The cross-reactivity with skeletal troponin T is therefore
<0.02% or lower (Table 2
).
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clinical evaluation
Healthy subjects.
The mean remission value for 64 healthy
individuals was 99.5% (SD = 0.6%). When this remission value was
converted into a concentration by the use of the established algorithm
for reader measurements, the respective cTnT value was <0.05 µg/L.
The visual reassessment of the test strip yielded only negative
results. The mean concentrations with the cTnT ELISA and CK-MB mass
were 0.00 µg/L (SD, 0.01 µg/L) and 1.35 µg/L (SD, 0.56 µg/L),
respectively.
Patients with chest pain but no clinical evidence for acute
myocardial ischemia.
The mean remission value for 325 blood
samples of 171 patients was 99.5% (SD, 1.4%), which corresponds to a
calculated concentration of <0.05 µg/L. The mean concentrations for
the cTnT ELISA was 0.01 µg/L (SD, 0.02 µg/L). The respective values
for CK activity and CK-MB mass were 47.2 U/L (SD, 42.4 U/L) and 1.36
µg/L (SD, 2.20 µg/L).
In six blood samples from three patients, the calculated cTnT concentrations were >0.1 µg/L (97% remission value). These tests were also positive when visual assessment of the rapid assay device was performed. When cTnT was measured with the ELISA technique, all but one (0.17 µg/L) sample had serum cTnT concentrations below 0.1 µg/L. Two of these samples had cTnT ELISA concentrations near the detection limit (0.08 µg/L). Clinical diagnosis was severe aortic valve stenosis in one case; in the other two cases, the diagnosis was known coronary artery disease but no evidence for acute ischemia based on cardiac enzymes and electrocardiogram (ECG).
Patients with definite AMI.
The mean remission value in the
351 samples from the 37 patients with definite AMI was 74.0% (SD,
21.9%), which corresponds to a calculated cTnT concentration of 4.67
µg/L. The mean concentration for the cTnT ELISA was 4.99 µg/L (SD,
9.36 µg/L). The respective values for CK activity and CK-MB mass
were 304.5 U/L (SD, 489.5 U/L) and 55.5 µg/L (SD, 140.2
µg/L).
Negative results were found in 21 of 351 measurements for the visual assessment of the rapid assay. When cTnT ELISA measurements were performed, results below 0.1 µg/L were found in 39 samples, and the mean concentration was 0.03 µg/L (SD, 0.03 µg/L). This number corresponded to the reader measurements (mean remission, 98.88%; SD, 1.5%). All samples with cTnT values <0.1 µg/L were obtained earlier than 5 h or later than 145 h after the onset of symptoms.
Patients with unstable angina pectoris.
The mean remission
value for patients with unstable angina pectoris was 97.8% (SD,
4.0%), which corresponded to a calculated reader cTnT
concentration of 0.14 µg/L. The mean concentration for the cTnT ELISA
was 0.09 µg/L (SD, 0.28 µg/L). The respective values for CK
activity and CK-MB mass were 45.6 U/L (SD, 66.1 U/L) and 1.78 µg/L
(SD, 2.82 µg/L).
One hundred twenty-six of 354 samples had cTnT values >0.1 µg/L with the ELISA technique, indicating the presence of minor myocardial damage. Ninety-four percent of the samples that were positive in the cTnT ELISA were also positive when the measurement was performed with the cardiac reader. In the visual reassessment, all 126 test strips were rated positive.
Kinetics of cTnT release in AMI.
The determination of cTnT in
sequential blood samples of AMI patients by the use of ELISA and the
cardiac reader produced similar time-dependent concentration changes
(Fig. 3
).
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Diagnostic performance in the early period of AMI.
The
diagnostic sensitivity and specificity of the quantitative bedside
assay were evaluated according to the time interval from the onset of
symptoms to analysis (Tables
3 and
4). Because of the debatable classification of
cTnT-positive patients with unstable angina, the groups were analyzed
both with (Table 3
) and without (Table 4
) the unstable angina patients.
Within the first 24 h, the diagnostic sensitivity was comparable
in all four diagnostic assays. The diagnostic specificities increased
for both cTnT determination methods to 100% when patients with
unstable angina pectoris were omitted from the analysis. As expected,
cTnT remained increased for longer time periods than CK and CK-MB.
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The areas under the ROC curves for analyses with and without patients
suffering from unstable angina pectoris are shown in Table 5
. Samples of different time intervals 0–24, 0–48, and 0–96 h
after onset of pain were evaluated. Again, similar performance of
measurements with the reader and the cTnT ELISA could be shown.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently it was shown that cTnT determinations may provide valuable information not only for better risk stratification but also for improved therapeutic decisionmaking in chest pain patients. The data of the FRISC trial convincingly showed that only cTnT-positive, but not cTnT-negative, patients with acute coronary syndromes benefit from low-molecular weight heparin (15). These findings may only be the beginning of a selected treatment of patients with acute coronary syndromes based on cTnT concentrations.
potential advantages of biochemical bedside testing
There are several potential advantages of bedside measurement of
biochemical markers with the new assay and reader device in patients
with chest pain: (a) A biochemical test result is
immediately available at the hands of the responsible physician and may
thus aid in the triage of chest pain patients, particularly if a
positive cTnT result is obtained. Even in university hospitals, the
time delay from venipuncture to the availability of a first result may
account for several hours. (b) There are many smaller
hospitals that do not provide 24-h stat biochemical testing. These
institutions may utilize a simple bedside test that can be quantified
conveniently by the developed reader. (c) The variability of
test results attributable to the reading of the test by untrained
personnel is excluded, and information about cTnT serum kinetics may be
obtained in consecutive testing of individual patients. (d)
Cardiac risk (9)(10) and therapeutic efficiency
(15) are influenced by the degree of the increase in cTnT.
It is therefore preferable to determine cTnT quantitatively. The
development of the quantitative test strip reader represents a further
advancement in bedside testing because a time-saving and sensitive
assay is now combined with a quantitative cTnT measurement.
(e) The reader can be linked to the hospital data management
system, and it will provide a numerical documentation of a quantitative
result. The reader printout can easily be included into the patient's
chart, taking care of the necessity of proper documentation.
analytical characteristics
Several substantial improvements of the previous test strip have
been implemented to provide a highly efficient testing of whole blood
samples for acute myocardial damage.
The antibody combination on the test strip now corresponds to the antibodies that are used in the cTnT ELISA system. This has produced a marked improvement in specificity of the test strip, reducing cross-reactivity with skeletal troponin T to <0.02%. Furthermore, the composition and quantitative relationship of dry chemistry constituents has been modified to ensure high sensitivity. Thus, even by visual assessment, the detection limit of the new test device has been reduced to 0.1 µg/L.
Analysis of the remission of the test strip signal demonstrated that the cardiac reader can detect cTnT concentrations below this cutoff value of 0.1 µg/L. The within-series imprecision and day-to-day imprecision studies yielded CVs of 9–14% and 15–18%, respectively. The method comparison showed a high correlation for concentrations of 0.1–3 µg/L; however, the accuracy was ± 15%. These values are acceptable, but clearly higher than those observed for the laboratory-based method. Thus, further improvements of the test relating to imprecision and accuracy are certainly needed. As a consequence, the measuring range of the reader was restricted in the final version to a concentration range of 0.1–3 µg/L; values between 0.05 and <0.1 µg/L are indicated as "low", values <0.05 µg/L are indicated as "negative".
When higher cTnT values are present in blood, dilution of the blood specimen would be required if a quantitative result is needed. Twelve minutes are needed for completion of the reading process. However, as soon as a positive line is detected on the test strip, the signal light on the cardiac reader is activated. Thus, therapeutic consequences can be taken before the quantitative result is printed by the reader.
In patients with AMI, unstable angina, and chest pain of noncardiac origin, this quantitative cTnT assay performs comparably to the more sophisticated laboratory-based assay. This also relates to the qualitative and quantitative recovery of cTnT at different days after the onset of AMI symptoms. With the rapid assay and the laboratory ELISA method, a similar time concentration curve was found in these patients.
More than 10 years ago, we first established myosin light chains and then troponin T as markers to overcome some of the limitations of biochemical testing for the detection of myocardial cell necrosis. With the development of the quantitative bedside assay for cTnT, we have completed these developments. As a result, it is now possible to measure within 12 min highly specific and sensitive cTnT without the dependency on a sophisticated clinical chemistry environment.
We do not believe that bedside testing and clinical chemistry testing are mutually exclusive methods. Clearly the laboratory-based method has a better analytical performance (4). Thus, if time is not critical and the appropriate equipment is available, the laboratory-based method may still be preferable. However, for many clinical situations and for many smaller hospitals, bedside testing may be the preferred method, particularly when the results are connected to the hospital data management system as is possible with the cardiac reader.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  B. Metzler, B. Haubner, E. Conci, J. Voelkl, J. Jehle, M. Bauer, D. Wolf, O. Pachinger, and Q. Xu Myocardial ischaemia-reperfusion injury in haematopoietic cell-restricted {beta}1 integrin knockout mice Exp Physiol, July 1, 2008; 93(7): 825 - 833. [Abstract] [Full Text] [PDF]
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 S. B. Rosalki, R. Roberts, H. A. Katus, E. Giannitsis, J. H. Ladenson, and F. S. Apple Cardiac Biomarkers for Detection of Myocardial Infarction: Perspectives from Past to Present Clin. Chem., November 1, 2004; 50(11): 2205 - 2213. [Abstract] [Full Text] [PDF]
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C. W. Hamm Cardiac Biomarkers for Rapid Evaluation of Chest Pain Circulation, September 25, 2001; 104(13): 1454 - 1456. [Full Text] [PDF]
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E. Giannitsis, M. Muller-Bardorff, S. Lehrke, U. Wiegand, R. Tolg, B. Weidtmann, F. Hartmann, G. Richardt, and H. A. Katus Admission Troponin T Level Predicts Clinical Outcomes, TIMI Flow, and Myocardial Tissue Perfusion After Primary Percutaneous Intervention for Acute ST-Segment Elevation Myocardial Infarction Circulation, August 7, 2001; 104(6): 630 - 635. [Abstract] [Full Text] [PDF]
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