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Articles |
1
Medizinische Klinik II, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany.
2
Abteilung Innere Medizin III, University of Heidelberg,
Heidelberg, Germany.
3
Boehringer Research Center, Tutzing, Germany.
4
Department of Clinical Chemistry, Lasarettet,
Helsingborg, Sweden.
a Author for correspondence. Fax 49/451/5006437; e-mail Medizinische Universit{at}t zu Lübeck.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key Words: indexing terms: cardiac markers • monoclonal antibodies • biotin–streptavidin interaction • creatine kinase
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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However, the first-generation troponin T ELISA (TnT 1) could also show falsely increased values in patients with severe skeletal muscle damage. These false-positive results may be explained by an unspecific binding of skeletal muscle troponin T to the wall of the test tube, which can then be detected by the cross-reactive enzyme-labeled antibody used in the TnT 1 assay. Obviously, this made TnT 1 results unreliable in patients with severe skeletal muscle injury, and we therefore decided to improve the specificity of the assay by replacing the cross-reactive antibody with a cardiac-specific monoclonal antibody (mAb). We here report on the development of this improved assay (TnT 2), its analytical characteristics, and its diagnostic performance.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reagents included with the newly developed Boehringer Mannheim Troponin T assay kit (TnT 2) are incubation buffer (40 mmol/L phosphate, pH 7.0), biotinylated anti-troponin T antibody M7 (1.5 mg/L), anti-troponin T antibody M11.7 labeled with horseradish peroxidase (>100 U/L), and five human cTnT calibrators (0–18.8 µg/L) in human serum matrix. Substrate buffer (100 mmol/L phosphate–citrate buffer, pH 4.4, containing sodium perborate, 3.2 mmol/L) and ABTS® substrate (2,2-azino-bis 3-ethylbenzothiazoline-6-sulfonate), 1.9 mmol/L, were also from Boehringer Mannheim.
preparatory procedures
Preparation of muscle homogenates.
Tissues from human
skeletal muscle and human myocardium were obtained postmortem from the
Department of Pathology no longer than 10 h after the patient's
death. Muscle tissue (100 g) was homogenized in 1 mL of a solution of
0.05 mol/L KCl, 0.05 mol/L Tris, 5 mmol/L EDTA, 1 mmol/L
dithiothreitol, and 2 KIU/L aprotinin (Trasylol), pH 7.0, with three
bursts of a homogenizer (Ultraturrax, Germany) at room temperature.
This first supernatant, containing the cytosolic proteins, was
recovered after centrifugation of the homogenate for 15 min at
14 000g. The remaining pellet was homogenized in 10 volumes
of a solution of 1.5 mol/L KCl, 0.05 mol/L Tris, 2 mmol/L EDTA, 5
mmol/L ATP, and 2 KIU/L aprotinin, pH 7.0, with three bursts of the
Ultraturrax homogenizer. The solution was stirred for 20 min at 4 °C
before centrifugation at 14 000g for 15 min. The resulting
second supernatant, containing the myofibrillar proteins, was also
saved. If not used immediately, both supernatants were stored as
aliquots at -20 °C.
Purification of troponin T.
The troponin T isoforms are
encoded by different genes and thus are proteins of differing amino
acid sequences. In the healthy human adult, the cTnT isoform is
expressed exclusively in the heart (14). Skeletal muscle
troponin T was prepared from human psoas muscle, and cTnT from cardiac
muscle tissue, by use of the same conventional techniques
(14)(15)(16) outlined previously (17). Tissue was
obtained from the Department of Pathology no more than 12 h
after the death of the patient. The molecular homogeneity of the
purified antigens was assessed by sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) according to
Laemmli (18). The specific absorption of troponin T at 280
nm was 0.706 L · g-1 · cm-1
(17).
Antibody selection and characterization.
Selection and
characterization of antibodies followed the protocols outlined in
detail previously (17). MAbs against human cTnT were
produced by somatic cell fusions of P3 x 63-Ag8–653 myeloma
cells and splenocytes from Balb/C mice immunized with four
intraperitoneal injections of purified human cTnT in Freund`s
adjuvant. The cell supernatants were screened by ELISA and the positive
cells were selected by a cell sorter (FACS; Becton Dickinson,
Heidelberg, Germany) and cloned. The cell clones selected by ELISA and
the FACS were grown in culture and screened again by ELISA for antibody
secretion (17). Cell clones secreting antibodies specific
for human cTnT were grown in culture or cryopreserved.
Characterization of the antibodies by Western blot.
Crude myofibrillar proteins and purified troponin T from human cardiac
and skeletal muscles were fractionated on a 9% SDS-PAGE
(18). The gels were stained with AgNO3 (1 g/L,
30 min) and developed with formaldehyde (15 mmol/L) plus
Na2CO3 (25 g/L). Immunoblotting was performed
as described by Towbin et al. (19). The proteins were
electrotransferred (100 mA, 50 V for 25 min) from the SDS-PAGE to
nitrocellulose membranes (Immobilon P membrane; Millipore, Waltham,
MA). The membranes were then blocked with 10 g/L bovine serum albumin
(BSA) solution. The troponin T antigen was identified by incubating the
membranes for 30 min at room temperature in phosphate-buffered saline
(PBS), pH 7.4, containing 2 µg/L anti-troponin T antibody and 50 g/L
BSA. Unbound antibodies were removed by washing with PBS. The membranes
were then incubated for 30 min at room temperature with
4000-fold-diluted goat-anti-mouse antibody labeled with peroxidase in
PBS containing 25 g/L BSA. The membranes were stained with 50 g/L
diaminobenzidine in the presence of 3 mL/L H2O2
for 3 min at room temperature (alternatively, with 0.025 mol/L
4-chloro-naphthol in PBS + 50 µL H2O2).
Purified human cTnT and proteins of known molecular mass were used as
controls.
assay development and characterization
Antibodies.
To determine the binding characteristics of
different pairs of antibodies with cTnT, we performed a biospecific
interaction analysis. The system we used for this (BIAcore-System;
Pharmacia Biosensor, Uppsala, Sweden) uses surface plasmon resonance to
detect real- time binding and dissociation of interacting molecules to
provide information on the affinity, specificity, kinetics, and
cooperativity of an interaction. The binding epitopes on the cTnT
molecule for the different antibodies chosen for assay development were
determined by testing the antibody binding to peptide fragments of cTnT
(as outlined in detail by Geysen et al. (20)) by using
12-amino acid peptides with a 1-amino-acid frameshift, starting from
the amino-terminus.
The biotinylization of the capture antibody (M11.7) was performed according to the method of Peters and Baumgarten (21) with the use of biotin cross-linked with N-hydroxysuccinimide. Horseradish peroxidase was coupled to the second mAb (M7) as outlined in detail before (17).
These two antibodies are applied in a one-step sandwich assay. The biotinylated capture antibody M11.7 binds with high affinity to the streptavidin-coated test tube. The signal antibody M7 is labeled with horseradish peroxidase. Addition of ABTS substrate produces a 405 nm absorbance signal that is proportional to the cTnT concentration, and the concentration is calculated by using Rodbard functions and dedicated software. As adapted to the ES 300 and 600 analyzers (Boehringer Mannheim), the turnaround time of this newly developed assay is 45 min.
Calibrators.
Five calibrators (purified bovine cTnT in
human serum) are used to generate the calibration curve. The new
calibrators have been measured by TnT 1. With the resulting preliminary
calibrator values a method comparison between TnT 1 and TnT 2 had been
performed. The difference between both methods expressed as percent of
deviation of the slope of the correlation curve had been calculated.
The preliminary calibrator values had been corrected by this
percentage.
Performance evaluation.
The specificity of
the assay was tested by serially diluting extracts of cytosolic
proteins from skeletal muscle and the troponin T purified from
diaphragm, psoas, and quadriceps muscle. The cross-reactivity was
calculated as the relative concentrations of skeletal troponin T and
cTnT at 50% maximal binding on the calibration curve.
The lower detection limit was determined by analyzing in single determination 21 blood samples from healthy volunteers. The mean concentration + 3SD for these samples was calculated.
Intraassay precision was assessed by measuring 10 times five serum samples with cTnT concentrations of 0.19, 5.14, 5.38, 9.39, and 13.74 µg/L. Day-to-day imprecision was determined by analyzing five serum samples of different cTnT concentrations (0.19, 0.30, 0.54, 5.28, and 14.89 µg/L) once each on 10 subsequent days. For each sample we calculated the mean ± SD concentration and CV.
other cardiac markers
Total CK activity was determined in the clinical chemistry
laboratory with a Clin Chem Analyzer (Miles, Tarrytown, NY) and the
reagents provided by the manufacturer. The upper reference limit for
total serum CK is 80 U/L in men and 75 U/L for women (25 °C). CK-MB
activity was determined by the immunoinhibition method (CK-MB-NAC;
Boehringer Mannheim). The upper reference limit for CK-MB activity at
25 °C is 10 U/L, or <5% of the total CK activity.
The TnT 1 assay was performed according to the manufacturer's instructions manual. Turnaround time for the TnT 1 is 90 min at room temperature. The discriminator value of cTnT used to indicate myocardial damage was >0.1 µg/L.
blood sampling
Blood (10 mL) was drawn into tubes and centrifuged, and the serum
samples obtained were stored at 4 °C. If analysis was not done
within 8 h, serum samples were stored at -20 °C. For all
AMI-suspected patients, the time from onset of pain to assay was
recorded. Serial blood samples were taken from each patient, on
admission and afterwards at 4–6-h intervals for the first 24 h,
at 8-h intervals on day 2, and one per day until day 14 or until
discharge. In patients without ischemic heart disease and patients with
skeletal muscle injury, one admission sample was drawn. We drew two
samples from 43 marathon runners, one before and one after the event.
subjects
We assayed samples from five groups of subjects. All gave written
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.
1. We compared TnT 1 and TnT 2 results in 323 samples from 47 AMI-suspected patients, an average number of 6.8 samples per patient having been collected during the hospital stay; slope, intercept, and Sey were calculated for the scatter plot.
2. To determine the reference range for cTnT measured with TnT 2, we assayed samples from 4955 patients suspected of endocrine disorders but without clinically apparent myocardial damage, who had been to ambulatory care units and offices of general practitioners.
3. The in vivo specificity of TnT 2 was tested in 43 healthy individuals after a 3-day marathon race and in 24 patients with rhabdomyolysis but without clinical evidence of myocardial damage.
4. Serum samples from AMI-suspected patients seen in the emergency room of the University Hospital in Heidelberg were used to assess the diagnostic performance of TnT 2. The final diagnoses in these patients were:
a) Chest pain but no evidence for acute cardiac disorders as assessed by history, clinical examination, ECG, echocardiography, and cardiac enzymes (47 serum samples, 47 patients). Additionally, the control group included 132 serum samples from 132 healthy volunteers.
b) Definite AMI (67 serum samples, 21 patients). The diagnostic criteria for definite AMI (WHO) were: monophasic ST-segment elevations of at least 0.2 mV in at least two adjacent leads of the 12-lead ECG with appearance of new Q-waves and reduction of R-waves and (or) a time-dependent change of serum activities of CK and CK-MB more than twice the upper limit of normal or an increased CK activity with a CK-MB fraction exceeding 5% of the total CK activity.
c) Possible (minor) myocardial cell damage (74 serum samples, 52 patients). These patients complained of either accelerating angina (Braunwald class I) or angina at rest (Braunwald class III) (22). AMI was excluded by ECG and results of cardiac enzyme measurements.
5. Patients with Duchenne muscular dystrophy (n = 20) and patients with end-stage renal disease undergoing chronic maintenance hemodialysis (n = 40).
statistical calculations
Data analyzed are given as either mean ± SD or as median
with 25th and 75th percentiles (quartiles). Method comparison was
performed as a scatter plot, showing slope, intercept, and Sey.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows the characterization of these cardiospecific mAbs (M11.7,
M1.16, M20.3) by West-ern blot analysis, in comparison with mAbs 1B10
and M7, which are used in the TnT 1 ELISA. MAb 1B10 binds not only to
the cardiac isoform of TnT and two of its degradation products but also
to the isoforms of troponin T expressed in psoas muscle. In contrast,
antibodies M7, M11.7, M1.16, and M 20.3 reveal no or only extremely
minor reactivity with skeletal troponin T from psoas muscle, quadriceps
muscle, and diaphragm or other myofibrillar proteins.
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The reactivity of the selected mAbs was further tested by ELISA with
purified troponin T isoforms from human and bovine cardiac or skeletal
muscle as antigens. MAbs M7 and M11.7 were less reactive with bovine
cTnT than with human cTnT. No species difference in reactivity was
observed with mAbs M20.3, M1.16, and 1B10. All but the 1B10 antibodies
cross-reacted by 
0.3% with the skeletal troponin T isoforms of psoas
muscle, quadriceps muscle, and diaphragm. Relative reactivity with
cardiac myosin light chains, troponin I, and tropomyosin was <0.1%.
The binding characteristics of different pairs of antibodies with cTnT were tested by biospecific interaction analysis with the BIAcore System. Antibody pairs M7–M11.7 and M7–M1.16 showed signals of 89% and 101%, respectively, relative to the antibody combination used in TnT 1 (M7–1B10 = 100%).
Locations of the respective epitopes of selected mAbs were mapped by a peptide scan of human cTnT. The epitopes of mAbs M7 and M11.7 are separated by only 6 amino acid residues, whereas the distance between the epitopes of mAbs M7 and M1.16 is 45 amino acid residues.
Figure 2
shows how much circulating cTnT was recovered by three cTnT
assays with the antibody combinations M7–M11.7, M7–M1.16, and
1B10–M7 (the combination used in TnT 1). Recovery of circulating cTnT
by the antibody combination M7–M1.16 was markedly less than that by
the 1B10–M7 and the M7–M11.7 combinations.
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assay characterization
The in vitro specificity (Fig. 3
) of the antibody combination M7–M11.7 was tested with use of
purified troponin T and homogenates from psoas muscle and cardiac
muscle as antigens. Using troponin T purified from human skeletal
muscle at concentrations as great as 1000 µg/L gave a
cross-reactivity <0.1%. When extracts of freshly prepared skeletal
muscle were tested in serial dilutions, no binding of the M7–M11.7
antibody combination was observed even at a 1:10 dilution. The same
data were obtained when homogenates from quadriceps muscle and
diaphragm or purified TnT from these tissues were tested.
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Detection limit.
The lower detection limit of TnT 2, as
assessed by 21 determinations of serum samples from healthy
individuals, was 0.011 or 0.012 µg/L (mean + 2SD or mean + 3SD,
respectively).
Calibration curves for TnT 1 and TnT 2 (Fig. 4
).
Fig. 4
shows the calibration curves for TnT 1 and TnT 2.
In TnT 2 the number of calibrators is reduced from 6 to 5. The slope
and the measuring range of both tests are comparable, because both
assays use a common cTnT calibrator.
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Intraassay precision.
The analysis of 10 determinations
of the same serum sample showed a CV 4.1% at all cTnT concentrations
tested. The mean ± SD concentrations measured, in µg/L (and
CV), were: 0.19 ± 0.01 (4.1%), 5.14 ± 0.06 (1.3%), 5.38
± 0.12 (2.2%), 9.39 ± 0.11 (1.2%), and 13.74 ± 0.1 (0.7%).
Day-to-day imprecision.
Between-day imprecision over the
whole range of the controls, 0.19–13.74 µg/L, was <5.8%. The mean
± SD concentrations, in µg/L (and CV), were: 0.19 ± 0.01
(5.8%), 0.3 ± 0.01 (3.8%), 0.54 ± 0.02 (4.5%), 5.28
± 0.17 (3.2%), and 14.89 ± 0.29 (2.0%).
Heparin plasma.
TnT results for heparin plasma
correlated well with those for serum (n = 95,
rs=0.97, P <0.005; data not shown).
Specificity in individuals with skeletal muscle injury.
In the 43 marathon runners, the median values (and 25th and 75th
percentiles) for troponin T determined by the new TnT 2 method and by
TnT 1 were 0.0 µg/L (0.0, 0.025) and 0.08 µg/L (0.03, 0.33),
respectively. The total CK activity in these subjects was 818 U/L
(546.5, 1107). The respective values for the 24 patients with
rhabdomyolysis were: TnT 2, 0.0 µg/L (0.0, 0.0); TnT 1, 3.07 µg/L
(2.04, 3.74); and CK activity, 14 820 U/L (3445, 32 200). All runners
and all patients with rhabdomyolysis had markedly increased activities
of total CK. As tested with the TnT 1, troponin T was >0.2 µg/L in
15 of 43 volunteers after their marathons and in 23 of the 24 patients
with rhabdomyolysis. No increase in troponin T (>0.1 µg/L) was
observed when the samples were tested by the newly developed TnT 2.
TnT 2 reference range.
The distribution of cTnT
concentrations in patients with nonacute cardiac diseases was analyzed
in serum samples from 4955 patients tested for possible endocrine
disorders in general practitioners' offices (Fig. 5
). cTnT was 0.02 µg/L in 80% of the patients, 0.04 µg/L
in 95% of the patients, 0.06 µg/L in 98.7% of the patients, and
0.1 µg/L in 99.6% of the patients. cTnT was 
0.1 µg/L in 21 of
the 4955 patients, exceeding 0.2 µg/L in 15; the highest
concentration measured was 1.02 µg/L.
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Method comparison: correlation between TnT 1 and TnT 2 in
AMI-suspected patients.
We compared TnT 1 with TnT 2 in 323
samples from 47 AMI-suspected patients (Fig. 6
). The slope, intercept, and Sey were 1.18, 0.01 µg/L, and
0.81 µg/L, respectively. For the lower range of values, 0–0.5 µg/L
(n = 117 serum samples), the values were 1.18, 0.004 µg/L, and
0.088 µg/L, respectively.
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clinical evaluation
Control group.
cTnT measurements by TnT 2 and total CK
activity of blood samples from healthy volunteers and patients without
any clinical evidence for cardiac ischemia are shown in Fig. 7
A. The median (and quartiles) concentration values measured in
132 healthy volunteers with the cardiac-specific TnT 2 were 0.0 µg/L
(0.0, 0.0), with the TnT 1 were 0.0 (0.0, 0.01), and total CK activity
was 37 U/L (26, 51). For the 47 patients without myocardial ischemia,
the respective values were: 0.0 µg/L (0.0, 0.03), 0.0 µg/L (0.0;
0.0), and 38 U/L (24.2, 50.5). CK activity was increased in 16 of these
latter patients (maximum 1235 U/L).
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Patients with AMI.
In the 67 samples from patients with
AMI (Fig. 7B
), the median (and quartiles) cTnT concentration measured
with the specific TnT 2 was 1.6 µg/L (0.245, 8.87) and with the TnT 1
was 1.57 µg/L (0.145, 9.3); CK was 118.5 U/L (47, 586.5). In 10 of 14
cases with increased cTnT but normal-range CK activity, the samples had
been obtained in the subacute phase of AMI, the other 4 samples having
been drawn early in AMI development. In 41 samples both CK and cTnT
were increased, rising to 20- and 100-fold the upper limit of normal,
respectively.
Patients with unstable angina.
In the 74 samples from
the patients with possible myocardial cell damage clinically classified
as unstable angina (Fig. 7C
), median (and quartiles) cTnT concentration
values measured with TnT 1 and TnT 2 were 0.035 µg/L (0.0, 0.2) and
0.07 µg/L (0.01, 0.25), respectively. CK activity in these samples
was 42.5 (25, 91.7) U/L.
In 35 of the 74 samples, cTnT was >0.1 µg/L, and CK activity exceeded the upper limit of normal in 14 of these 35 samples. In 5 of the 7 patients with an increased CK but normal cTnT, the coronary angiography was normal, suggesting a noncardiac origin of CK in these patients.
Renal failure and muscular dystrophy patients.
We tested
40 serum samples from patients with renal failure undergoing chronic
hemodialysis. Median cTnT concentration values (and quartiles) were
0.07 µg/L (0.027, 0.25) by TnT 1, 0.09 µg/L (0.06, 0.13) by TnT 2.
The concentrations were above normal (>0.2 µg/L) by TnT 1 in 11 of
the 40 patients and in 5 by TnT 2.
Serum samples from 20 patients with muscular dystrophy gave median (and quartiles) cTnT concentration values of 0.54 µg/L (0.13, 1.8) by TnT 1 and 0.1 µg/L (0.02, 0.18) by TnT 2. Concentrations were >0.2 µg/L by TnT 1 in 14 of the 20 patients and in 4 by TnT 2. CK values were 1986 U/L (632, 3214), being above normal in all but 1 patient.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When we and others analyzed patients with more extensive skeletal muscle injuries and CK activities in blood exceeding 25-fold the upper limit of normal, cTnT increases were observed in some individuals without clinical findings of acute myocardial cell necrosis (7)(17). Our hypothesis regarding these troponin T increases is that they may have been false-positive results attributable to an unspecific binding of skeletal muscle troponin T to the test tube and detection by the cross-reactive mAb 1B10 used as label.
To avoid these false-positive results, we developed a new, more-specific, version of the Troponin T ELISA utilizing a new antibody combination (two cardiospecific mAbs). Great care was taken in the selection of a suitable new pair of mAbs, to find antibodies that would be more specific than but at least as sensitive as the former pair. Selection for specificity was carried out by Western blotting and ELISA of purified cardiac and skeletal troponin T as well as homogenates of human myocardium and of different human skeletal muscles such as diaphragm, psoas, and quadriceps muscle.
The binding capacity of different pairs of antibodies was determined with a biospecific interaction analysis (BIAcore). The mAb pairs with the highest relative response were selected for further investigation.
Various pairs of high-affinity antibodies were checked for their recovery of troponin T in patients' blood. Antibody combination M7–M11.7 showed a recovery similar to that of antibody combination M7–1B10, used in TnT 1 assay; M7–M1.16 detected only 50% of circulating cTnT.
The epitope-mapping showed that the binding epitopes of antibody combination M7–M11.7 are only 6 amino acid residues apart, whereas combination M7–M1.16 binds epitopes ~45 amino acid residues apart. Possible cleavage by proteases between the binding epitopes of mAbs M7 and M1.16 might explain the decreased detection of circulating troponin T by this antibody combination. The number of "sandwiches" formed and the resulting signal would accordingly be diminished. We therefore used the antibody combination M7–M11.7 for further assay development. In TnT 2, mAb M11.7 is coupled to biotin and mAb M7 is labeled with horseradish peroxidase.
The in vitro specificity analysis showed no signal for troponin T purified from skeletal muscle, even at concentrations as great as 1000 µg/L. No false-positive results were obtained when we tested 43 healthy marathon runners and 24 patients with severe skeletal muscle damage but no evidence for acute cardiac disorders.
The highly specific TnT 2 assay detected above-normal TnT concentrations in some patients with chronic renal failure (5 of 40 patients tested) or muscular dystrophy (4 of 20 patients tested). That is, the proportion of cTnT-positive patients is less when measured with the TnT 2 assay than with the TnT 1 assay, indicating that some of the previously reported positive cTnT results may result from an unspecific binding. The reason for cTnT increases in the patients with muscular dystrophy and the patients on maintenance hemodialysis remains to be determined and is the subject of ongoing clinical investigations. Some have suggested that cTnT may be reexpressed in regenerating rat or human skeletal muscle (25)(26). Alternatively, the increased troponin T may reflect true myocardial damage because dilative cardiomyopathy may result from muscular dystrophy (27) and a substantial proportion of patients with end-stage renal failure suffer from severe coronary artery disease (29), [30]. In a separate investigation on 105 patients on maintainance hemodialysis, we observed a significant positive correlation between the presence of coronary artery disease and increased cTnT concentrations (Haller et al., in preparation for publication).
The time-dependent concentration changes of troponin T in patients with
AMI and the number of positive troponin T results in patients with
unstable angina or AMI as measured in patients` blood by the new TnT 2
show good agreement with the results by the TnT 1 over a wide
concentration range. Thus, the diagnostic sensitivities of the first-
and second-generation troponin T assays appear to be equivalent.
Moreover, the improved specificity of the new assay provides sufficient
reproducibility to be useful in clinical practice (intra- and
interassay CVs of 4.1% and 5.8%, respectively).
The detection limit of the new assay, defined as 3SD above the mean absorbance at 405 nm for 21 determinations of blood samples from healthy volunteers, is 0.0123 µg/L—another improvement over TnT 1 (detection limit 0.04 µg/L). In addition, the turnaround time could be reduced from 90 min for TnT 1 to 45 min for TnT 2.
Testing troponin T concentrations in a large number of patients with endocrine disorders allowed us to set the clinical discriminator value for troponin T at 0.1 µg/L. This discrimination value correctly classified 99.6% of the patients with endocrine disorders as having normal heart function. However, if the few patients with troponin T >0.2 µg/L are considered to be true-positive patients with unrecognized acute cardiac disorders, the proportion of patients classified correctly at a discriminator value of 0.06 and 0.1 µg/L would be 99% and 99.8%, respectively. Prospective study of an unselected group of chest pain patients may thus be necessary to clarify the clinical significance of troponin T values between 0.06 and 0.1 µg/L. Preliminary data from the Fragmin in Acute Ischemic Syndrome (12) trial indicate an increasing risk for cardiac events in patients with unstable angina and non-Q-wave AMI, beginning at troponin T concentrations of 0.06 µg/L. The GUSTO II investigators also noted a linear increase of cardiac risk with increasing troponin T concentrations (10). The GUSTO II and other investigators, however, used a 0.1 µg/L discriminator value in their clinical trials on patients with acute ischemic syndromes and efficiently stratified their patients according to risk by using this discriminator value.
In summary, this second-generation ELISA for troponin T is substantially improved in specificity, allowing the differentiation of cardiac and skeletal muscle damage even in patients with severe skeletal muscle injury. At the same time, the turnaround time of the assay could be reduced to 45 min without loss in analytical precision and clinical sensitivity. Therefore, we expect that TnT 2 will replace TnT 1 in clinical practice.
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Footnotes |
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References |
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 A. S. Jaffe, L. Babuin, and F. S. Apple Biomarkers in Acute Cardiac Disease: The Present and the Future J. Am. Coll. Cardiol., July 4, 2006; 48(1): 1 - 11. [Abstract] [Full Text] [PDF]
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 L Tulloh, D Robinson, A Patel, A Ware, C Prendergast, D Sullivan, and L Pressley Raised troponin T and echocardiographic abnormalities after prolonged strenuous exercise--the Australian Ironman Triathlon Br. J. Sports Med., July 1, 2006; 40(7): 605 - 609. [Abstract] [Full Text] [PDF]
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 L. Babuin and A. S. Jaffe Troponin: the biomarker of choice for the detection of cardiac injury Can. Med. Assoc. J., November 8, 2005; 173(10): 1191 - 1202. [Abstract] [Full Text] [PDF]
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K. Arakawa, Y. Kawai, T. Kumamoto, N. Morikawa, M. Yoshida, H. Tada, R. Kawaguchi, K. Taniguchi, I. Miyamori, Y. Kominato, et al. Serum deoxyribonuclease I activity can be used as a sensitive marker for detection of transient myocardial ischaemia induced by percutaneous coronary intervention Eur. Heart J., November 2, 2005; 26(22): 2375 - 2380. [Abstract] [Full Text] [PDF]
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 M. Rajappa and A. Sharma Biomarkers of Cardiac Injury: An Update Angiology, November 1, 2005; 56(6): 677 - 691. [Abstract] [PDF]
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K. Hayashida, N. Kume, T. Murase, M. Minami, D. Nakagawa, T. Inada, M. Tanaka, A. Ueda, G. Kominami, H. Kambara, et al. Serum Soluble Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 Levels Are Elevated in Acute Coronary Syndrome: A Novel Marker for Early Diagnosis Circulation, August 9, 2005; 112(6): 812 - 818. [Abstract] [Full Text] [PDF]
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 R. Rej Clinical Chemistry through Clinical Chemistry: A Journal Timeline Clin. Chem., December 1, 2004; 50(12): 2415 - 2458. [Abstract] [Full Text] [PDF]
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C. Heeschen, C. W. Hamm, V. Mitrovic, N.-H. Lantelme, H. D. White, and for the Platelet Receptor Inhibition in Ischemic S N-Terminal Pro-B-Type Natriuretic Peptide Levels for Dynamic Risk Stratification of Patients With Acute Coronary Syndromes Circulation, November 16, 2004; 110(20): 3206 - 3212. [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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A. S. Jaffe and H. Katus Acute Coronary Syndrome Biomarkers: The Need for More Adequate Reporting Circulation, July 13, 2004; 110(2): 104 - 106. [Full Text] [PDF]
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 B L Norgaard, K Andersen, K Thygesen, J Ravkilde, P Abrahamsson, L Grip, and M Dellborg Long term risk stratification of patients with acute coronary syndromes: characteristics of troponin T testing and continuous ST segment monitoring Heart, July 1, 2004; 90(7): 739 - 744. [Abstract] [Full Text] [PDF]
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 A. S. Gami, A. Svatikova, R. Wolk, E. J. Olson, C. J. Duenwald, A. S. Jaffe, and V. K. Somers Cardiac Troponin T in Obstructive Sleep Apnea Chest, June 1, 2004; 125(6): 2097 - 2100. [Abstract] [Full Text] [PDF]
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 G. Carr-White, T. Koh, A. DeSouza, E. Haxby, M. Kemp, J. Hooper, D. Gibson, and J. Pepper Chronic stable ischaemia protects against myocyte damage during beating heart coronary surgery Eur. J. Cardiothorac. Surg., May 1, 2004; 25(5): 772 - 778. [Abstract] [Full Text] [PDF]
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M. Herrmann, J. Scharhag, M. Miclea, A. Urhausen, W. Herrmann, and W. Kindermann Post-Race Kinetics of Cardiac Troponin T and I and N-Terminal Pro-Brain Natriuretic Peptide in Marathon Runners Clin. Chem., May 1, 2003; 49(5): 831 - 834. [Full Text] [PDF]
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J Oldgren, L Wallentin, L Grip, R Linder, B.L Norgaard, and A Siegbahn Myocardial damage, inflammation and thrombin inhibition in unstable coronary artery disease Eur. Heart J., January 1, 2003; 24(1): 86 - 93. [Abstract] [Full Text] [PDF]
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B. J. Freda, W. H. W. Tang, F. Van Lente, W. F. Peacock, and G. S. Francis Cardiac troponins in renal insufficiency: Review and clinical implications J. Am. Coll. Cardiol., December 18, 2002; 40(12): 2065 - 2071. [Abstract] [Full Text] [PDF]
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 C. Lowbeer, A. Gutierrez, S. A. Gustafsson, R. Norrman, J. Hulting, and A. Seeberger Elevated cardiac troponin T in peritoneal dialysis patients is associated with CRP and predicts all-cause mortality and cardiac death Nephrol. Dial. Transplant., December 1, 2002; 17(12): 2178 - 2183. [Abstract] [Full Text] [PDF]
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E. J. Fransen, J. H. C. Diris, J. G. Maessen, W. Th. Hermens, and M. P. van Dieijen-Visser Evaluation of "New" Cardiac Markers for Ruling Out Myocardial Infarction After Coronary Artery Bypass Grafting Chest, October 1, 2002; 122(4): 1316 - 1321. [Abstract] [Full Text] [PDF]
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 R. J. Aviles, A. T. Askari, B. Lindahl, L. Wallentin, G. Jia, E. M. Ohman, K. W. Mahaffey, L. K. Newby, R. M. Califf, M. L. Simoons, et al. Troponin T Levels in Patients with Acute Coronary Syndromes, with or without Renal Dysfunction N. Engl. J. Med., June 27, 2002; 346(26): 2047 - 2052. [Abstract] [Full Text] [PDF]
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E. M. Antman Decision Making with Cardiac Troponin Tests N. Engl. J. Med., June 27, 2002; 346(26): 2079 - 2082. [Full Text] [PDF]
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S. Fredericks, J. F. Murray, N. D. Carter, A. M.S. Chesser, S. Papachristou, M. M. Yaqoob, P. O. Collinson, D. Gaze, and D. W. Holt Cardiac Troponin T and Creatine Kinase MB Content in Skeletal Muscle of the Uremic Rat Clin. Chem., June 1, 2002; 48(6): 859 - 868. [Abstract] [Full Text] [PDF]
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S. Fredericks, J. F. Murray, M. Bewick, R. Chang, P. O. Collinson, N. D. Carter, and D. W. Holt Cardiac Troponin T and Creatine Kinase MB Are Not Increased in Exterior Oblique Muscle of Patients with Renal Failure Clin. Chem., June 1, 2001; 47(6): 1023 - 1030. [Abstract] [Full Text] [PDF]
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A. Hammerer-Lercher, P. Erlacher, R. Bittner, R. Korinthenberg, D. Skladal, S. Sorichter, W. Sperl, B. Puschendorf, and J. Mair Clinical and Experimental Results on Cardiac Troponin Expression in Duchenne Muscular Dystrophy Clin. Chem., March 1, 2001; 47(3): 451 - 458. [Abstract] [Full Text] [PDF]
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J. C.J.M. Swaanenburg, B. G. Loef, M. Volmer, P. W. Boonstra, J. G. Grandjean, M. A. Mariani, and A. H. Epema Creatine Kinase MB, Troponin I, and Troponin T Release Patterns after Coronary Artery Bypass Grafting with or without Cardiopulmonary Bypass and after Aortic and Mitral Valve Surgery Clin. Chem., March 1, 2001; 47(3): 584 - 587. [Full Text] [PDF]
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Y. Sato, T. Yamada, R. Taniguchi, K. Nagai, T. Makiyama, H. Okada, K. Kataoka, H. Ito, A. Matsumori, S. Sasayama, et al. Persistently Increased Serum Concentrations of Cardiac Troponin T in Patients With Idiopathic Dilated Cardiomyopathy Are Predictive of Adverse Outcomes Circulation, January 23, 2001; 103(3): 369 - 374. [Abstract] [Full Text] [PDF]
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B. Lindahl, H. Toss, A. Siegbahn, P. Venge, L. Wallentin, and The FRISC Study Group Markers of Myocardial Damage and Inflammation in Relation to Long-Term Mortality in Unstable Coronary Artery Disease N. Engl. J. Med., October 19, 2000; 343(16): 1139 - 1147. [Abstract] [Full Text] [PDF]
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D. Wayand, H. Baum, G. Schatzle, J. Scharf, and D. Neumeier Cardiac Troponin T and I in End-Stage Renal Failure Clin. Chem., September 1, 2000; 46(9): 1345 - 1350. [Abstract] [Full Text] [PDF]
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 M. T. Rademaker, V. A. Cameron, C. J. Charles, E. A. Espiner, M. G. Nicholls, C. J. Pemberton, and A. M. Richards Neurohormones in an ovine model of compensated postinfarction left ventricular dysfunction Am J Physiol Heart Circ Physiol, March 1, 2000; 278(3): H731 - H740. [Abstract] [Full Text] [PDF]
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M Lund, J.K French, R.N Johnson, B.F Williams, and H.D White Serum troponins T and I after elective cardioversion Eur. Heart J., February 1, 2000; 21(3): 245 - 253. [Abstract] [PDF]
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S. L. Braun, A. Barankay, and D. Mazzitelli Plasma Troponin T and Troponin I after Minimally Invasive Coronary Bypass Surgery Clin. Chem., February 1, 2000; 46(2): 279 - 281. [Full Text] [PDF]
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V. Ricchiuti and F. S. Apple RNA Expression of Cardiac Troponin T Isoforms in Diseased Human Skeletal Muscle Clin. Chem., December 1, 1999; 45(12): 2129 - 2135. [Abstract] [Full Text] [PDF]
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 D. B Sacks Acute coronary ischemia: troponin I and T Vascular Medicine, November 1, 1999; 4(4): 253 - 256. [Abstract] [PDF]
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C. Lowbeer, A. Ottosson-Seeberger, S. A. Gustafsson, R. Norrman, J. Hulting, and A. Gutierrez Increased cardiac troponin T and endothelin-1 concentrations in dialysis patients may indicate heart disease Nephrol. Dial. Transplant., August 1, 1999; 14(8): 1948 - 1955. [Abstract] [Full Text] [PDF]
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 E. H. Herman, J. Zhang, S. E. Lipshultz, N. Rifai, D. Chadwick, K. Takeda, Z.-X. Yu, and V. J. Ferrans Correlation Between Serum Levels of Cardiac Troponin-T and the Severity of the Chronic Cardiomyopathy Induced by Doxorubicin J. Clin. Oncol., July 1, 1999; 17(7): 2237 - 2237. [Abstract] [Full Text] [PDF]
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M. Muller-Bardorff, T. Rauscher, M. Kampmann, S. Schoolmann, F. Laufenberg, D. Mangold, R. Zerback, A. Remppis, and H. A. Katus Quantitative Bedside Assay for Cardiac Troponin T: A Complementary Method to Centralized Laboratory Testing Clin. Chem., July 1, 1999; 45(7): 1002 - 1008. [Abstract] [Full Text] [PDF]
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A. H.B. Wu, F. S. Apple, W. B. Gibler, R. L. Jesse, M. M. Warshaw, and R. Valdes Jr. National Academy of Clinical Biochemistry Standards of Laboratory Practice: Recommendations for the Use of Cardiac Markers in Coronary Artery Diseases Clin. Chem., July 1, 1999; 45(7): 1104 - 1121. [Abstract] [Full Text] [PDF]
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C. W. Hamm, C. Heeschen, B. Goldmann, A. Vahanian, J. Adgey, C. M. Miguel, W. Rutsch, J. Berger, J. Kootstra, M. L. Simoons, et al. Benefit of Abciximab in Patients with Refractory Unstable Angina in Relation to Serum Troponin T Levels N. Engl. J. Med., May 27, 1999; 340(21): 1623 - 1629. [Abstract] [Full Text] [PDF]
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B. L. Norgaard, K. Andersen, M. Dellborg, P. Abrahamsson, J. Ravkilde, K. Thygesen, and for the TRIM study group Admission risk assessment by cardiac troponin T in unstable coronary artery disease: additional prognostic information from continuous ST segment monitoring J. Am. Coll. Cardiol., May 1, 1999; 33(6): 1519 - 1527. [Abstract] [Full Text] [PDF]
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T W Koh, G S Carr-White, A C DeSouza, F D Ferdinand, J Hooper, M Kemp, D G Gibson, and J R Pepper Intraoperative cardiac troponin T release and lactate metabolism during coronary artery surgery: comparison of beating heart with conventional coronary artery surgery with cardiopulmonary bypass Heart, May 1, 1999; 81(5): 495 - 500. [Abstract] [Full Text]
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F. Van Lente, E. S. McErlean, S. A. DeLuca, W. F. Peacock, J. S. Rao, and S. E. Nissen Ability of troponins to predict adverse outcomes in patients with renal insufficiency and suspected acute coronary syndromes: a case-matched study J. Am. Coll. Cardiol., February 1, 1999; 33(2): 471 - 478. [Abstract] [Full Text] [PDF]
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F. S. Apple, R. H. Christenson, R. Valdes Jr., A. J. Andriak, A. Berg, S.-H. Duh, Y.-J. Feng, S. A. Jortani, N. A. Johnson, B. Koplen, et al. Simultaneous Rapid Measurement of Whole Blood Myoglobin, Creatine Kinase MB, and Cardiac Troponin I by the Triage Cardiac Panel for Detection of Myocardial Infarction Clin. Chem., February 1, 1999; 45(2): 199 - 205. [Abstract] [Full Text] [PDF]
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L. Holmvang, M. S. Luscher, P. Clemmensen, K. Thygesen, and P. Grande Very Early Risk Stratification Using Combined ECG and Biochemical Assessment in Patients With Unstable Coronary Artery Disease (A Thrombin Inhibition in Myocardial Ischemia [TRIM] Substudy) Circulation, November 10, 1998; 98(19): 2004 - 2009. [Abstract] [Full Text] [PDF]
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V. Ricchiuti, E. M. Voss, A. Ney, M. Odland, P. A. W. Anderson, and F. S. Apple Cardiac troponin T isoforms expressed in renal diseased skeletal muscle will not cause false-positive results by the second generation cardiac troponin T assay by Boehringer Mannheim Clin. Chem., September 1, 1998; 44(9): 1919 - 1924. [Abstract] [Full Text] [PDF]
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D. S. Ooi and A. A. House Cardiac troponin T in hemodialyzed patients Clin. Chem., July 1, 1998; 44(7): 1410 - 1416. [Abstract] [Full Text] [PDF]
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O. Hetland and K. Dickstein Cardiac troponins I and T in patients with suspected acute coronary syndrome: a comparative study in a routine setting Clin. Chem., July 1, 1998; 44(7): 1430 - 1436. [Abstract] [Full Text] [PDF]
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 L. Coudrey The Troponins Arch Intern Med, June 8, 1998; 158(11): 1173 - 1180. [Full Text] [PDF]
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A. H. B. Wu, Y.-J. Feng, R. Moore, F. S. Apple, P. H. McPherson, K. F. Buechler, G. Bodor, f. t. A. A. for, and C. C. S. o. c. Standardization Characterization of cardiac troponin subunit release into serum after acute myocardial infarction and comparison of assays for troponin T and I Clin. Chem., June 1, 1998; 44(6): 1198 - 1208. [Abstract] [Full Text] [PDF]
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O. Hetland and K. Dickstein Cardiac Troponin T by Elecsys System and a Rapid ELISA: Analytical Sensitivity in Relation to the TropT (CardiacT) ""Bedside"" Test Clin. Chem., June 1, 1998; 44(6): 1348 - 1350. [Full Text] [PDF]
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C. Haller, J. Zehelein, A. Remppis, M. Muller-Bardorff, and H. A. Katus Cardiac troponin T in patients with end-stage renal disease: absence of expression in truncal skeletal muscle Clin. Chem., May 1, 1998; 44(5): 930 - 938. [Abstract] [Full Text] [PDF]
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A. Lavoinne and G. Hue Serum Cardiac Troponins I and T in Early Posttraumatic Rhabdomyolysis Clin. Chem., March 1, 1998; 44(3): 667 - 668. [Full Text] [PDF]
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M. S. Luscher, K. Thygesen, J. Ravkilde, and L. Heickendorff Applicability of Cardiac Troponin T and I for Early Risk Stratification in Unstable Coronary Artery Disease Circulation, October 21, 1997; 96(8): 2578 - 2585. [Abstract] [Full Text]
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H. Baum, S. Braun, W. Gerhardt, G. Gilson, G. Hafner, M. Muller-Bardorff, W. Stein, G. Klein, C. Ebert, K. Hallermayer, et al. Multicenter evaluation of a second-generation assay for cardiac troponin T Clin. Chem., October 1, 1997; 43(10): 1877 - 1884. [Abstract] [Full Text] [PDF]
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M. D. McLaurin, F. S. Apple, E. M. Voss, C. A. Herzog, and S. W. Sharkey Cardiac troponin I, cardiac troponin T, and creatine kinase MB in dialysis patients without ischemic heart disease: evidence of cardiac troponin T expression in skeletal muscle Clin. Chem., June 1, 1997; 43(6): 976 - 982. [Abstract] [Full Text] [PDF]
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P. O. Collinson To T or Not to T, That Is the Question Clin. Chem., March 1, 1997; 43(3): 421 - 423. [Full Text] [PDF]
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C. Heeschen, C. W. Hamm, U. Laufs, S. Snapinn, M. Bohm, and H. D. White Withdrawal of Statins Increases Event Rates in Patients With Acute Coronary Syndromes Circulation, March 26, 2002; 105(12): 1446 - 1452. [Abstract] [Full Text] [PDF]
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