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Articles |
1
Institut für Klinische Chemie und Pathobiochemie, Klinikum rechts der Isar der TU München, Ismaningerstr. 22, D-81675 München, Germany.
2
Institut für Laboratoriumsmedizin, Deutsches
Herzzentrum, München, Germany.
3
Department of Clinical Chemistry, Lasarettet
Helsingborg, Sweden.
4
Laboratoire de Biochimie–Immunopathologie, Centre
Hospitalier de Luxembourg.
5
Abteilung für Klinische Chemie und
Laboratoriumsmedizin der Universität Mainz, Mainz, Germany.
6
Medizinische Klinik II, Medizinische Universität
Lübeck, Lübeck, Germany.
7
Abteilung für Laboratoriumsmedizin/Klinische
Chemie, Krankenhaus St. Georg, Hamburg, Germany.
8
Boehringer Mannheim, Mannheim, Germany.
a Author for correspondence. Fax + 49 89 4140–4875; e-mail baum{at}mail.klinchem.med.tu-muenchen.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the clinical usefulness of the high diagnostic sensitivity of cTnT in cardiac diseases is well established, the diagnostic specificity of cTnT has been doubted by various findings. The first version of the cTnT assay (TnT 1) had a cross-reaction of at least 2% with skeletal muscle (9). This did not interfere with the diagnostic specificity in the coronary care unit (CCU) in the absence of skeletal muscle damage, but obviously made the results difficult to interpret in traumatized patients. cTnT increases have been observed in certain types of myositis such as sclerodermatomyositis and Duchenne disease (10), and in patients with chronic or acute renal failure (11)(12)(13)(14) without evidence of ischemic myocardial injury with the TnT 1 assay. Even the cardiospecificity of the analyte itself has been questioned by the observation that cTnT determined with the TnT 1 assay may be reexpressed in regenerating skeletal muscle (15). Whether these findings are due to the described cross-reactivity with skeletal muscle TnT, expression of the cardiac isoform of TnT in diseased muscles, or to MMD not detectable with other clinical methods or biochemical markers is as yet unclear. To assure the analytical cardiospecificity, an improved "second-generation" assay with a different antibody panel, Enzymun® TnT (TnT 2), has been developed (16).
The present multicenter study was carried out to evaluate the cardiospecific TnT 2 assay and compare it with TnT 1 in different patient groups.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) participated, following a general protocol with some
individual deviations described in the text.
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analytical methods
The TnT 1 assay was performed with two different lots, ELISA TnT
lots 186190 and 186145. The TnT 2 assay (Enzymun Troponin T) has two
monoclonal antibodies. A new cardiospecific monoclonal antibody
(11–7) was developed to serve as the biotinylated capture
antibody (16), and the cardiospecific capture antibody of
the TnT 1 assay (17) is now used as the signal antibody.
The assay time has been reduced to 45 min on the ES system and the
stability of the reagents and calibrators has been extended to 2 weeks
(incubation solution) and 1 week at +4 °C, respectively. All
measurements of cTnT were made with first- and second-generation assays
on ES 300 or ES 700 analyzers (Boehringer Mannheim).
The evaluation was carried out with two lots, A and B. The calibrator values of lot A were reassigned after the completion of the study to guarantee comparable results of both lots.
Imprecision.
Three control sera (controls A, B, and C)
and different human serum pools were assayed for intraassay (20
samples/run) and interassay (each day up to 43 days) imprecision.
Analytical range and linearity.
To determine the
analytical sensitivity, all collaborating laboratories measured the
zero calibrator supplied with the reagent kit in 20 replicates. Mean +
2SD of the signal were calculated and read of the actual calibration
curve to give the lower detection limit. Linearity was investigated by
serial dilution of different sera containing high amounts of cTnT. The
measured values in terms of recovery were plotted against the expected
cTnT values derived from linear regression analysis.
Stability of cTnT.
Short-term and long-term stability
studies were performed. We aliquoted and tested 10 serum samples from
patients with cTnT values between 0.11 and 15.34 µg/L. TnT results in
fresh serum were compared with the results after different times of
storage up to 3 months at -20 °C. We also tested the influence of
repeated freezing and thawing.
Collection tubes.
To study the influence of different
serum and plasma collection tubes, we collected from five patients
different serum and plasma specimens: white (without anticoagulation),
brown (without anticoagulation, with gel), red (K2EDTA),
blue (NH4-heparin), green (sodium citrate), yellow
(Na-fluoride) tubes, all from Sarstedt; serum with gel barrier from
Terumo and from Becton Dickinson (type SST). Also, at two sites a more
significant number of paired values for heparin plasma and serum
(n = 46 and n = 18) and on one site for citrate plasma and
serum (n = 19) was collected (all tubes from Sarstedt).
Method comparison.
We compared the TnT 1 and the TnT 2
assays using 1084 serum specimens from randomly selected patients with
increased CK activity. For each different testing site and for the
combined groups, regression analysis was performed and comparisons were
made.
clinical studies
We compared TnT 1 and TnT 2 in several clinical populations to
look for differences in the diagnostic sensitivity and specificity.
Subjects.
We compared the diagnostic sensitivity of the
two assays in serum samples from patients with ischemic heart diseases:
341 serum samples from patients with acute myocardial infarction (AMI),
130 from patients with unstable angina pectoris, and 51 from patients
undergoing percutaneous transluminary coronary angioplasty (PTCA). To
investigate possible unspecificity in patients with skeletal muscle
disease or renal diseases, we examined 33 serum samples from patients
with Duchenne disease, serum samples before and after a race from 42
marathon runners, and serum samples from 30 patients with end-stage
renal failure undergoing chronic hemodialysis. Furthermore, we compared
TnT 1 and TnT 2 results in the following individual cases: a case with
extensive hypoxic skeletal muscle injury and increased total CK, a
rhabdomyolysis in hereditary myoglobulinuria, a progressing
dermatomyositis, a pressure-induced skeletal muscle injury, and a
decubitus case.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. CVs between 1.4% and 5.9% for intraassay imprecision and
between 2.4% and 9.5% for interassay imprecision were obtained over
the whole measuring range and the highest CVs for the control serum in
the range of the cutoff.
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Analytical sensitivity.
The lower detection limit was
calculated to be 0.01 µg/L at evaluation sites 2 and 5 and 0.02
µg/L at site 7. A functional sensitivity of 20% interassay CV was
estimated between 0.05 and 0.1 µg/L.
Linearity.
Recovery of the expected concentration for
one typical dilution experiment is shown in Fig. 1
. The assay seems sufficiently linear over the whole measuring
range.
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Interfering substances.
Serial dilutions of sera
containing 0.21 and 0.32 µg/L cTnT were diluted with sera containing
possible interfering substances. No interference was seen (±10%) for
hemoglobin up to 0.62 mmol/L, bilirubin up to 1128.6 µmol/L, and
lipemia up to 17.1 mmol/L triacylglycerol.
Analyte stability.
cTnT values in fresh samples and in
samples stored at room temperature up to 24 h, at +4 °C over 1
week, and at -20 °C for 3 months show no significant deviation
(evaluator 7). A regression analysis for the 10 samples gives the
following equations according to Passing/Bablok (x =
fresh, y = stored):
1 day room temperature: y = 0.89x + 0.02
1 week +4 °C: y = 0.95x + 0.01
3 months -20 °C: y = 0.91x + 0.01
At a different evaluation site no loss of cTnT concentration after 5 days of storage at room temperature was found (site 8).
Effect of sampling material.
The analyte
concentrations obtained from eight different sample collection tubes of
five patients each do not show any significant influence by tube
material; 14 of 40 cTnT values were found from a 95% confidence
interval of the mean, but the deviation was not indicated as
significant in any case when calculated by a statistics program
(Astute; DDU Software). Regression analysis of the serum/plasma
comparisons at three evaluation sites show slightly lower concentration
values for heparin plasma (without volume correction) and no
significant differences for citrate plasma (after volume correction)
compared with serum (x = plasma, y =
serum):
Lab. 2: Heparin plasma y = 1.10x + 0.01, r = 0.99 (n = 18)
Lab. 5: Citrate plasma y = 1.01x + 0, r = 1.00 (n = 19)
Lab. 7: Heparin plasma y = 1.07x + 0, r = 0.96 (n = 46)
Analytical comparison with TnT 1.
Results are
summarized in Table 3
. The slopes of the separate evaluation of the lower range
deviate obviously from those of the whole range. This deviation is
pronounced for lot A with preliminary standardization and reduced
for lot B with final standardization. The relation between TnT 1 and
TnT 2 can be influenced by the selection of the sample material as can
be seen in the slope differences of the individual testing sites, which
used patient samples with various clinical backgrounds. Fig. 2
shows a characteristic example for the relation between the
first- and second-generation assays in the whole analytical range (Fig. 2a
) and in the lower range (Fig. 2b
) based on the final standardization
lot B with samples preferably from a CCU. A considerable number of sera
show cTnT values <0.1 µg/L with TnT 2 and >0.1 µg/L with TnT 1,
thus indicating some possible unspecificity of the first-generation
assay.
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clinical comparisons
Marathon runners.
As shown in Fig. 3
, no cross-reactivity with skeletal muscle isoforms of TnT in
marathon runners can be detected with TnT 2.
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Muscular dystrophy.
In patients with Duchenne disease,
increased values are significantly reduced with TnT 2 (Fig. 4
). If related to a decision concentration of 0.2 µg/L, which
may be taken for safety reasons in situations outside of the CCU, 8 of
33 samples show slightly increased concentrations with TnT 2; the
highest value was found at 0.33 µg/L. In contrast, 25 of 33 samples
show values >0.2 µg/L with TnT 1, and the range extends up to 13.5
µg/L.
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Individual cases with potential cross-reaction of skeletal
muscle troponin T.
One case of pure skeletal muscle damage caused
by two severe epileptic seizures showed a 12-fold increase of total CK
activity and 33-fold of TnT 1, but no increase at all of TnT 2,
confirmed by no clinical or electrocardiogram (ECG) signs of myocardial
damage (Fig. 5
a). In a case with severe hypoxic skeletal muscle damage after
embolism in the foot, we found an increase of total CK and TnT 1, and a
late increase of CK-MB mass; however, we saw a TnT 2 concentration at
the detection limit in all examined samples (Fig. 5
b). A case of
hereditary myoglobulinuria with severe rhabdomyolysis, 90-fold
increased myoglobin, 200-fold increased total CK activity, 13-fold
increased CK-MB mass, and 1.3-fold increased creatinine showed a TnT 2
value of 0.07 µg/L. A case of prednisolone-treated progressing
dermatomyositis with 95-fold increased total CK activity and sevenfold
increased CK-MB mass showed a TnT 2 value of 0.02 µg/L. A decubitus
case with 125-fold increased total CK and increasing creatinine showed
a TnT 2 value of 0.02 µg/L.
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Patients with renal insufficiency.
Only small
differences between TnT 1 and TnT 2 were seen in patients with renal
failure (Fig. 6
). With TnT 2 in six (20%) of 30 patients (median of the 30
patients: 0.1 µg/L, range 0–5 µg/L), cTnT is detectable above the
safer decision concentration of 0.2 µg/L, compared with 11 (37%) of
30 patients (median 0.13 µg/L, range 0–6.92 µg/L) with TnT 1.
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Patients with ischemic heart disease.
A good correlation
between TnT 1 and TnT 2 was found in patients with AMI, unstable angina
pectoris, or undergoing PTCA (Table 3
). The differences in the slopes
of the regression analysis can be explained mainly by standardization
effects as mentioned above.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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On the basis of the investigation of ~5000 noncardiac diseased and healthy individuals, Müller-Bardorff et al. (16) reported that the cutoff value for the clinical decision of a myocardial damage can be maintained at 0.1 µg/L as already specified for the first-generation assay. However, this value seems to have an increased reliability because several unspecific increases can be excluded.
The crucial criteria for the assessment of TnT 2 compared with TnT 1 are the cardiosensitivity and cardiospecificity. To investigate sensitivity, we compared both assays in patients with ischemic myocardial injury, AMI, or unstable angina pectoris and undergoing PTCA. In all patients, no significant differences between the two assays were observed. These findings show that both assays have the same sensitivity in patients with ischemic myocardial injury, give the same clinical information, and are interconvertible with each other without loss of evidence in longitudinal observations.
The cardiospecificity has to be shown in very different clinical situations. If the clinical background of a patient is rather unclear or characterized by multimorbidity, clinical decision making should be based on a raised cTnT cutoff value of 0.2 µg/L. For the first-generation cTnT assay, Hafner et al. (11), Bhayana et al. (12), and Li et al. (13) reported increased cTnT values without evidence of ischemic myocardial damage in patients with end-stage renal failure undergoing maintenance hemodialysis. Baum et al. (14) demonstrated increased first-generation assay cTnT values also in patients with acute renal failure, with sepsis, and after liver transplantation.
Regarding the results in 30 patients with end-stage renal failure
undergoing chronic hemodialysis in the present study, the portion of
increased values obtained with the second-generation assay is only
slightly reduced from 37% to 20%—if related to the decision
concentration of 0.2 µg/L—in comparison with the first-generation
assay. A more significant reduction of pathologically increased values
with TnT 2 can be observed in the patient group of Duchenne disease,
and the eight pathological cTnT concentrations of 33 samples are only
marginally above the limit of 0.2 µg/L. A more distinct improvement
in the cardiospecificity of TnT 2 is found in the patient groups with
typical skeletal muscle damage. An assessment of specificity can be
based here on the usual "cardiac" cutoff of 0.1 µg/L. Twenty of
42 marathon runners who showed increased cTnT values after a race with
TnT 1 had no increased values at all with TnT 2. In the case of purely
skeletal muscle damage (Fig. 5
), assuming a 2% cross-reactivity of TnT
1 with skeletal muscle TnT and a detection limit of 0.02 µg/L for TnT
1, peak skeletal muscle TnT would have been 165 µg/L, and
correspondingly cross-reactivity of TnT 2 must be <0.02/165 =
0.01%. Thus, for all practical purposes, interference by skeletal
muscle TnT cross-reactivity can be excluded for the second-generation
assay. The results of the first-generation assay are explainable, as
this assay shows cross-reactivity of at least 2% with the skeletal
muscle isoform of TnT (9) due to the fact that only one
cardiospecific monoclonal antibody has been used in this assay. In the
new assay the capture and label antibodies are both cardiospecific
anti-cTnT antibodies without cross-reactivity with the skeletal muscle
isoform. This was confirmed with Western blot analysis
(16). Increases therefore cannot simply be explained by
possible cross-reactivity with the skeletal muscle isoform of TnT.
The increased cTnT values in patients with end-stage renal failure and Duchenne disease therefore indicate either possible minor myocardial injury not detectable with "classical" biochemical markers for the detection of myocardial damage or expression of the cardiac isoform of TnT in diseased muscles. As a history of ischemic myocardial damage is expected to occur more often in hemodialysis patients (19)(20), minor ischemic myocardial damage without clinical signs cannot be ruled out. MMD in patients with Duchenne disease can also not be ruled out with standard criteria. These two possibilities as a source of cTnT must be investigated in further studies. Up to that point, misinterpretation can be avoided by the simple means of determining cTnT in two samples, e.g., with 6-h intervals. Myocardial damage will cause a significant rapid increase; this does not occur by purely renal failure or muscular disease.
In conclusion, this modified assay for cTnT measurement is superior over the first-generation assay, with an increased specificity and a reduced turnaround time, but without a change in sensitivity in the diagnosis of AMI or MMD.
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Acknowledgments |
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Footnotes |
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References |
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, TnT 1; 
, TnT 2; 
, total CK (ECCLS, 37 °C); 
, CK-MB
mass (IMX). (a) Cardiospecific TnT 2 remained at zero
excluding myocardial damage (evaluation site 3). (b)
Increases could be caused by skeletal muscle, myocardial damage, or a
combination of both. In contrast, TnT 2 remaining at the detection
limit in all nine samples ruled out myocardial damage in agreement with
absence of clinical or ECG signs (evaluation site 3).