CLIN. CHEM. 40/6,
900-907
(1994)
#{149}
Enzymes
and Protein
Markers
Cardiac Troponin-T Immunoassay for Diagnosis of Acute Myocardial Infarction
H. B. Wu,”6 Roland Valdes, Jr.,2 Fred S. Apple,3 Terrie Gornet,4
Mayfleld-Stokes,2 Ann Marie Ingersoll-Stroubos,3 and Barbara Wiler5
Alan
We evaluated the analytical and clinical performance of
an immunoassay for cardiac troponin T (cTnT). Within-run
and total imprecision ranged from 1.6 to 11.3%. The sensitivity and linear range was 0.015 and 13 g/L, respectively. Frozen samples were stable for at least 8 weeks.
No interferences were seen with lipids or bilirubin (total
and conjugated). Hemoglobin caused a negative bias at
concentrations >4 gIL. Heparinized plasma showed a 6%
negative bias compared with serum. The clinical utilityof
cTnT was compared with that of creatine kinase (CK)-MB
(mass assay). The sensitivityof cTnT measurements from
63 patients with acute myocardial infarction (AMI) (cTnT
cutoff 0.1 g/L) was 60% at 0-3 h, 59% at 3-6 h, 94% at
6-9 h, 90% at 9-12 h, 99% at 12-24 h, 92% at 24-48 Ii,
83% at 48-72 h, and 100% at 72-96 h. Corresponding
results for CK-MB (cutoff 5.0 pg/L and 2.5% relative index) were 45%, 64%, 82%, 97%, 87%, 81%, 54%, and
59%. The specificity of the markers from 49 non-AMI
patients was 46% and 79% for cTnT and CK-MB, respectively. We show that CK-MB is more specificfor diagnosis
of AMI, and propose that cTnT is more sensitive to myocardial injury.
Indexing
Terms: sandwich immunoassay/creatine kinase isoenzymes/serum cardiac marker/skeletal muscle injury/receiver-operating characteristic curves
Creatine
kinase
(EC 2.7.3.2)
isoenzyme
MB
(CK-MB)
in serum is the most widely used marker for the diagnosis of acute myocardial
infarction
(AM!) (1, 2). The
development
of automated
immunoassays
for CK-MB
has led to improvements
in analytical sensitivity
and
turnaround
time (2-4). Clinical studies
have shown
that assay of cardiac troponin may challenge the role of
CK.MB in diagnosis of AM! (5-10). Cardiac troponin T
(cThT) is a 37-kDa polypeptide
subunit of the myofibrilJar regulatory troponin complex (11, 12). It is one of the
three major types (troponin-C,
-I, and -T) found in striated muscle.
Since most of the troponin
complex
is
1Departments
of Pathology and Laboratory Medicine, Hartford
Hospital, Hartford, CT.
2Uthversfty of Louisvffle Hospital, Louisville, KY.
3Hennepin County Medical Center, Minneapolis, MN.
4University
of Texas Medical School, Houston, TX.
5Boehringer
Mannheim Corporation, Indianapolis,
IN.
6Author for correspondence. Fax 203-545-5206.
7Nonstandard
abbreviations: cTnT, cardiac troponin-T; cTnl,
cardiac tropornn-I; smTnT, skeletal muscle troponin-T; CK-MB,
creatine kinase isoenzyme MB; AMI, acute myocardial infarction;
%RI, percent relative index; HCMC, Hennepin County Medical
Center; ULH, University of Louisville Hospital; UTMSH, University of Texas Medical School, Houston; EKG, electrocardiogram;
ROC, receiver-operating
characteristic.
Received August 16, 1993; accepted March 10, 1994.
900
CUNICAL CHEMISTRY, Vol. 40, No. 6, 1994
Mary
Ann Stone,2 Stephanie
bound to contractile
elements,
a prolonged
release of
troponin following AM! is expected because of the destruction and clearance
of structural
elements
from
muscle cells. However, a small amount
of troponin
is
present free in the cytoplasm,
and this maybe released
within
the first few hours after cell injury (13).
Assays for measuring
serum concentrations
of cTnT
(8-10, 13-16) and cardiac troponin-!
(cTnI) (6, 8, 17)
have been reported; research assays for cTnT have led to
the development
of an automated 90-mm immunoassay
(16). Clinical trials conducted in Europe have reported
the use of cTnT for the detection of AM! (7, 9, 10, 16).
These studies either used earlier versions of the cTnT
assay (9) or analyzer (ES22) (5, 10), or compared results
with CK-MB by immunoinhibition
(5, 9, 10).
The purpose of this multicenter
study was to evaluate
the analytical and clinical performance of the cThT assay on the ES 300 for imprecision,
transferability,
detection limit, linearity,
sample
stability,
interferences,
and specimen
collection requirements.
We determined
the reference range and verified a cutoff concentration
for cTnT in serum for detecting
myocardial
injury. The
clinical sensitivity
and specificity
of the cThT assay for
diagnosis of AM! was compared with the Stratus!! mass
assay
of CK-MB.
Assays
involving
cardiac-specific
monoclonal
antibodies to cThT show promise because
such antibodies
exhibit little or no cross-reactivity
with
skeletal
muscle troponin
(18). Thus patients with skeletal muscle disease or injury, in whom serum CK-MB
concentrations
are increased,
should not have high and
potentially
interfering
concentrations
of cardiac troponm (19).
Materials and Methods
Analytical Study
Testing sites. Three laboratory
sites were included
in
this study. The primary and tertiary care hospitals
were
Hennepin
County Medical Center (HCMC), Minneapolis, MN; University of Louisville
Hospital
(ULH), Louisville, KY; University
of Texas Medical School (UTMSH), Houston, TX. The study was coordinated
by
Boehringer
Mannheim,
Indianapolis,
!N. The same protocol was followed at all sites. Transferability
studies
were conducted at ULH, UTMSH,
and Boehringer
Mannheim. A study of blood collection tubes was performed only at HCMC. This protocol was reviewed and
approved by Institutional
Review Boards at each study
site.
Analytical
methods.
All measurements
of cTnT were
made with an enzyme immunoassay
on the ES300
(CardiAC
Troponin
T, Boehringer
Mannheim)
with
streptavidin-coated
tubes (16). This assay involves two
antibodies.
The capture antibody
(M7) is
for cTnT; the enzyme-labeled
(horseradish
peroxidase) antibody (1B1O) has a 12% cross-reactivity
with skeletal
muscle troponin T (smTnT). Although
these antibodies were raised against human cTnT, they
also cross-react with bovine and rabbit cardiac tissue
because of the high degree of interspecies homology (20).
The calibration curve was constructed
from 6 calibrators
ranging from 0 to 13 tg/L. Calibrators were prepared
from purified bovine troponin T and put into a human
serum protein matrix. The assay requires two on-instrument incubation
steps of 60 and 25 mm at 25#{176}C.
For the clinical
studies, CK-MB was measured
by a
mass assay on the Stratus I! analyzer (Baxter Healthcare, Miami, FL). Total CK was measured
with the
Ektachem
700RX (Eastman Kodak, Rochester, NY) at
HCMC, and with the Hitachi 717 analyzers (Boehringer
Mannheim)
at ULH and UTMSH. The percent relative
index (%RI) was calculated as: [CK-MB (pgfL)/total CK
(U/L)] x 100. Each of these assays was in routine use at
these respective
institutions.
Quality control was performed according to current procedures at these hospitals. The reference ranges for total CK (U/L), CK-MB
(g/L), and R! (%) were 40-200, 0-7, and 0-2.0, respectively, at HCMC; 19-205 (males), 17-111 (females), 0-5,
and 0-3 at ULH; and 83-200,
0-7, and 0-2.5 at UTMSH. We considered these differences in the reference
range to be negligible,
considering
the range of values
observed after AM!, and used a single limit for CK-MB
of 0-5.0 pgfL and 0-2.5% RI.
Imprecision
and transferability.
A modified “midi”
EP3-T protocol was performed (21). For the external
sites, three pools (low, medium, and high) from patients
and one commercial
control (Precitrol-N,
Boehringer
Mannheim)
were assayed six times (24 samples/run)
each day for 10 days (240 total samples).
To assess
transferabifity,
we assayed 19 samples of fresh human
serum at ULH and Boehringer Mannheim,
and lOsamples at ULH, UTMSH, and Boehringer Mannheim,
using the same lot of reagents. We also assayed identicallot (lyophilized)
kit controls at each clinical site.
Analytical sensitivity, linearity, and recovery. To dotermme
the lowest detectable concentration,
we measured the within-run
(n = 20) precision of the absorbance measurement
of the zero calibrator supplied with
the reagent
kit. Detection
limits were defined as the
mean absorbance
values plus 3 SD relative
to their
respective
concentrations
on the calibration
curve. To
assess linearity,
we diluted a serum sample containing
a
high cTnT concentration
with the zero calibrator supplied by the kit. Measured
cTnT concentrations
were
plotted against expected cTnT values, and a linearity
bias plot was computed
to indicate the goodness-of-fit
to
a straight
line in terms of residual percent recovery
from expected linearity. For recovery, to a serum sample
containing
no cThT, we added human serum from a
pooled sample containing
a high concentration
of cThT
to final concentrations
ranging
from -3 to 7 pgfL. Percent recovery was computed as the measured
value/
expected
value
x 100.
monoclonal
specific
Stability
of cTnT. Both short-term
and long-term
studies of sample
stability
were performed.
We allquoted and tested 12 serum samples from different patients in which cTnT concentrations
ranged from 0.46 to
6.9 gfL. The tests were conducted
at various time intervals
up to 194 h after storage of the samples at 25#{176}C,
2-8#{176}C,
and -20#{176}C.
cTnT results were plotted vs time of
storage. A limit of ± 10% was used to assess stability. To
assess long-term stability
we stored samples
from 40
patients with initial cTnT concentrations
of 0.04-6.45
gfL for 3-8 weeks at -20#{176}C.
Results after storage were
compared with measurements
obtained before storage.
Using
SDs from our precision studies, we used acceptance criteria of <2 SD for samples with cThT concentrations <1.0 jgfL, and <3 SD for samples 1.0
gfL.
Interference studies. Various
interferents
were added
to serum samples containing
a measured
concentration
of cThT. The interferents
were as follows: !ntralipid
(Cutter
Labs., Berkeley, CA) was added to produce lipid
concentrations
up to 12.5 g/L; a hemolysate
prepared
from heparinized
blood was added to produce hemoglobin concentrations
up to 10 g/L; unconjugated
bilirubin
(Fluka,
Buchs, Switzerland)
dissolved
in 0.1 mol/L
NaOH was added to produce bilirubin concentrations
up
to 645 mgIL; and an icteric serum sample
containing
158 mgfL conjugated
bilirubin
(total bilirubin
208 mgfL)
and no cTnT was added to produce bilirubin
concentrations up to 13 g/L. A limit of ± 10% of the unsupplemented serum sample was used.
Plasma
collection tubes. At one site (HCMC), nine
patients
presenting
to the coronary care unit had blood
drawn into four different collection tubes: red (no anticoagulant),
green (lithium heparin,
14.4 USP mU/L);
purple (K2EDTA, 1.5 g/L), and blue (sodium citrate, 25.8
mmo]JL),
all from Beckton Dickinson (Rutherford,
NJ).
These tubes were used to assess the effects of anticoagulant interference
in the assay; a 95% confidence
(P
<0.05) interval was used for comparing differences.
Reference range. We randomly
selected and equally
distributed
112 healthy ambulatory
subjects from each
hospital site for the reference range study. These subjects were deemed free of clinical
complications
or medications on the basis of interviews.
CK-MB in these
samples was measured
by immunoassay.
The reference
range for cThT was determined
from the central 95% of
values from this population.
Many of these individuals
were employees of the participating
hospitals.
Clinical
Study
Subjects. We obtained
serial
blood samples
from 63
patients diagnosed
by attending
physicians
at the time
of discharge as having had AM!. The diagnosis, based on
criteria established by the World Health Organization,
included typical or atypical
chest pain, unequivocal
changes in the electrocardiogram
(EKG), and increased
activity of CK and concentrations
of CK-MB on serial
blood collections
(22). !n the absence of EKG evidence
and a consistent clinical history,
diagnosis
of AM! may
have been based solely on increases
in total CK and
CK-MB. The concentration
of cTnT was not made
CLINICAL CHEMISTRY, Vol. 40, No. 6, 1994
901
known
to attending
and was not used in
The onset of chest pain was noted
in all patients
included in this study. Patients were
excluded if the time of onset was not known or not
documented
in the record. We also collected 116 serial
blood samples
from 49 patients with chest pain in whom
a diagnosis of AMI was ruled out.
Sample collection. All samples
collected
for this study
were ordered by attending or resident physicians as part
of the management
of patients; no additional
samples
were drawn solely for the purposes of this study. Informed consent was therefore deemed unnecessary
by
the Institutional
Review Boards at the study sites. Samples were grouped into time intervals
after the onset of
chest pain: 2-6, 6-12, 12-24, 24-48, and 48-96 h. All
were collected into evacuated
red-top tubes containing
no preservatives.
The blood was allowed to clot and was
centrifuged
before analysis. Total CK and CK-MB were
assayed within 8 h after collection and stored at 4#{176}C.
For
cTnT, samples were stored at 4#{176}C
for up to 72 h or frozen
at -20#{176}C
for up to 3 weeks before batch analysis. For
AM! patients, samples were collected at regular intervals for up to 7 days from presentation
in the emergency
department.
For non-AM! patients, samples were collected on the first and second day after admission.
We
prepared receiver-operating
characteristic
(ROC) curves
for cThT vs CK-MB at various
time intervals after the
onset of chest pain.
physicians
making the diagnosis.
C
c’J
cJ
3
0
0
C,
C
.0
I-
0
Cl,
.0
0
1
2
3
4
6
6
7
8
9
10
11
12
13
Cardiac TnT (pg/L)
Fig.1. Calibration curves for cTnT obtained at three independent
laboratory sites: (U) UTMSH, (S) ULH, and (A), HCMC.
Three calibration curves from one site (ULH)and two from the others, all from
a single lotof reagent are shown. Panel A Is the expanded portion of the low
concentration area from panel B. A spilne lit is used for each curve.
Results
AnalyticalStudy
Assay calibration.
In Fig. 1 we show several calibration curves obtained
from all three sites. These data
demonstrate
the consistency
in range of absorbance
values and response slopes between laboratory sites and for
several individual assay runs from each site. The curves
selected (of 12 from each site) represent the beginning,
middle, and end of the 90-day evaluation
period. The
curves for the low concentrations
are expanded
in Fig.
l#{192}
to contrast the reproducibifity
of the standards between sites.
Imprecision
and transferability.
Table 1 shows representative
data for within-run
and total imprecision
at
one of the testing sites. The values are within
limits
established by the manufacturer.
Results of split samples demonstrated
good transferability
of results between sites (BMC (y) vs ULH (x), y = 1.037x + 0.008
r = 0.999, n = 21; and UTMSH
(y)vs ULH (x),y =
1.050x
0.055 p.gfL, r = 0.991, n = 10). The relative
accuracy between
laboratory
sites was also tested with
controls provided with each reagent kit and assayed
once during each run of the project. Mean results
from
the three laboratories
for two levels of controls were
0.14, 0.17, and 0.17 ,ug/L for the low control, and 4.81,
4.60, and 4.71 g/L for the high control.
Analytical
sensitivity,
linearity, and recovery. To determine
the lowest detection limits, we calculated the
average
absorbance
values (SD) for the zero calibrator
(n = 20): 0.0153 (0.0011),
0.0194 (0.0017), and 0.0066
(0.0010) g/L for the HCMC, ULH, and UTMSH sites,
-
902
CLINICAL CHEMISTRY, Vol. 40, No. 6, 1994
Table 1. RepresentatIve
ImprecisIon
wIthin-run and total
of cTnT.
Control pools
Mean, iiaJL
WithIn-run CV, %‘
Low
0.003
112
Medium
3.65
4.2
High
7.39
4.1
Commercial control
0.40
8.5
a Averageof 10 within-runtrials, n
6 for each trial.
b
= 60
Total,
%b
166
5.2
4.6
11.3
respectively.
Corresponding
detection limits computed
from these data were 0.0187, 0.0246, and 0.0007 pgfL,
respectively,
with an average of 0.0 15 g/L. For linearity, the plots of expected concentrations
of cTnT (y) vs
measured values (x) produced a regression
curve of y =
1.OOx
0.0004 zgfL, r = 0.997, n = 30, for the three
sites combined. These results show that the cTnT assay
is linear up to at least 13 gfL. With a sensitivity
of
0.0 15, the dynamic
range of the assay covers more than
three orders of magnitude.
The recovery
values for a
blank serum sample supplemented
with high cThT were
93%, 100%, and 101% for expected cThT concentrations
of 3.84, 6.33, and 6.90 pg/L, respectively.
Stability
of cTnT. In Fig. 2 we show the percent recovery from the starting concentration
for all samples
combined for the three temperatures
tested.
cTnT is
stable for at least 194 h at -20#{176}C
and 146 h at 2-6#{176}C.
Stability
at room temperature
is lost after 24 h. For
long-term
stability,
38 of 40 samples
(95%) met our
criteria for stability
when stored at -20#{176}C
for 8 weeks.
-
Sample Stability
105
11o
>..
.100
a,
>
o
‘l0o,
C.,
890
0
90
cc
280
85
-U--
25
-#{149}-
2-6#{176}
80
--
-20#{176}
15
70
5
an
0
2
4
#{149}
6
24
48
72
#{149}
169
#{149}
8
#{149}
407
Bilirubin
146194
#{149}
#{149}
645
(mg/L)
Time (Hours)
Fig.2. Stability of cTnT in serum stored at 25#{176}C
(#{149}),
2-6#{176}C
(#{149}),
and
-20#{176}C
(A), measured on the ES-300.
Each data point is the mean of 12 separate patients’ specimens measured at
each of the threelaboratories (symbols as inFig. 1). The two horizontalilnes
are the
±
10% recovery relative to first measurement at time zero.
Interference
the
>#{176}
0)
>
0
C.)
0)
interference
studies.
studies
In Fig. 3 we show the results
with Intralipid,
of
hemoglobin,
and total (unconjugated) direct bilirubin.
Only hemoglobin at concentrations
>4 g/L demonstrated
interference
in the cTnT assay. For direct bilirubin at concentrations
of 53, 79, 105, and 132 mgfL, the recovery
values for
cTnT were 100%, 98%, 100%, and 102%, respectively
(data not plotted), indicating no significant interference.
Plasma collection tubes. To determine
the acceptability of drawing
blood in different blood collection tubes,
we collected blood from nine patients at HCMC into
each of the four different tubes. Mean (SD) results for
blood collected in heparin,
EDTA, citrate, and no proservatives
(serum) were 4.52 (6.55), 5.67 (7.26), 4.20
(5.58), and 4.82 (6.15) gfL, respectively.
A >5% average deviation
of recovery
compared
with serum
was
observed with all the anticoagulants
used. At-test comparison of means relative to serum produced
results oft
= 0.94,
1.86, and 2.52 for heparin,
EDTA, and citrate,
respectively
(t <2.306 was required for P = 0.05). Thus,
heparin had the lowest deviation compared with serum.
Reference range. All normal subjects had CK-MB values that were within the reference range established
at
each institution.
The results for cThT are shown in Fig.
4 and indicate a skewed distribution.
Using the central
95%, we calculated
the reference range to be 0-0.08
jJL. This is within the cutoff limit of 0.10 pgfL recommended by the manufacturer
for diagnosis of myocardial injury.
ClinicalStudy
Diagnostic
efficiency for AMI. Table 2 shows the data
for cThT and CK-MB for all the patients studied. For
AM! patients, the data are further grouped according
to
the reported time of onset of chest pain. In Fig. 5, ROC
curves are presented that indicate the clinical sensitivity and specificity at different cutoff limits. Fig. 6 shows
results with single cutoff limits of 0.1 pg/L for cTnT and
a combined cutoff of 5.0 pgfL and 2.5% RI for CK-MB.
These data show that during the critical first 6 h after
cc
0
0
110
I.;
105.
>.
100.
>
o
C.)
90.
95.
85.
80.
0
2.5
5
Intralipid
7.5
10
12.5
(gIL)
Fig. 3. Interference plot of cTnT measured on ES-300.
Bilirubin (A), hemoglobin (B), and Intralipid (C) were added toserum samples
at the concentrations indicated. % recovery is calculated relative to Initialvalue
without potential Interferent added. Symbols are as in Fig. 1; 4) is the average
of the three laboratory sites.
onset of chest pain, the clinical sensitivity
for cTnT and
CK-MB are roughly equivalent.
The sensitivity
for
CK-MB is highest between 9 and 48 h, after which time
values begin to decrease. In contrast, the sensitivity
for
cTnT remains high 96 h after onset. Fig. 7 shows mean
results for all patients in the study.
The clinical specificities
of cThT and CK-MB in 49
patients in whom the diagnosis of AMI was ruled out
were 46% and 79%, respectively.
Increasing
the cutoff
concentration
for cTnT to 0.2 p.g/L improved the specificity to 54%, as shown in Fig. 5. Retrospective
reviews
of the medical
records
indicated
that many of the patients that were positive for cTnT and negative
for
CK-MB had multiple illnesses
besides
cardiovascular
disease, such as pneumonia, liver disease, stroke, cancer, and cardiac failure.
CLINICALCHEMISTRY,Vol. 40, No. 6, 1994
903
405 nm, with the reaction rate being influenced by nonspecific adherence and absorbance of hemoglobin or red
cell constituents
remaining
after the washing
procedure. In any event, slight to moderate hemolysis (hemoglobin <4 g/L) did not cause interference.
Bilirubin
and
lipids did not interfere with this assay.
Serum
samples
from 112 healthy
individuals
had
cTnT values 0.1
p.g/L; most (96%) had <0.05 pgfL.
Although
such concentrations
produce an upper reference limit of 0.08 gfL, we used a decision limit of 0.1
pgfL for the clinical studies. A higher cutoff concentration of 0.2 gJL was used in studies performed in Europe
(5,16). Our cutoff limit was selected to optimize clinical
sensitivity
to minor myocardial
injury.
An objective of the clinical study was to determine
if
cThT was effective for diagnosis of AMI during the first
6 h after onset of chest pain. Release
of cTnT from the
cytosolic pool may enable earlier detection of AM! than
other cardiac markers if a low cutoff concentration
is
used. However, our results, showed a clinical sensitivity
of only 63% from 0-6 h for cTnT, which is insufficient for
effective
early diagnosis.
These findings are in agreement with those of Mair et al. (10), who reported a 57%
sensitivity
for emergency
department
patients
(mean
time of presentation
4 h); Katus eta!. (9), who reported
a 50% sensitivity
for patients presenting within 3 h; and
Bodor et al. (17), who reported sensitivities
of 17% between 0 and 4 h and 77% between 4 and 8 h after chest
pain for cTnI.
Our results also confirm other reports that cTnT remains abnormally
increased in serum for a much longer
time after onset of AM! than does CK-MB. This corresponds to the gradual release of proteins
due to degradation of structural
elements (10, 11, 17, 23). The cTnT
assay will eventually
obviate the need for assays
of
lactate dehydrogenase
isoenzymes.
The dinical specificity
of 46% reported
in this study is
much lower than in previous reports. Bodor et a!. found
cTnI sensitivities
of 78% and 83% in patients with and
without chronic ischemic
cardiac
disease,
respectively
(17). Gerhardt
et al. reported a 98% specificity,
although
some non-AM!
cases with minor myocardial
damage
80
‘70
6O
a,
g40
U-
8
__1
0
0.01
0.02
I
I
0.03
-
6
0.04
2
1
2
2
0.05
0.08
0.09
0.1
Cardiac TnT (pg/L)
Fig. 4. Distribution of cTnT values measured in serum from 112
healthy adults on the ES-300.
Numbers on bars are the number of subiects with values at the cTnT concentrations indicated.
DIscussIon
This study describes the first evaluation
of a cTnT
assay performed
on the ES300 analyzer
in the US. The
imprecision
studies
for cTnT from three
laboratories
demonstrated
CVs that compare well with other reports
(5, 10, 13, 16). Between sites, the day-to-day reproducibffity of controls gave consistent values with no drifts
or shifts, demonstrating
assay transferability.
The linear range for the cTnT assay was broad. Of serum samples obtained
from AMI or non-AMI patients,
<10%
required
dilution. The absorbance response of the calibration curve within the concentration
range of the calibrators was reproducible
between sites (Fig. 1). Dilution of serum
specimens
gave linear recoveries
of
expected cThT values throughout a wide concentration
range, sufficient to meet clinical needs reported by others (16, 17). With regard to plasma samples,
only tubes
containing
heparin
produced
results
comparable
to
those obtained
with serum. We did not attempt to explain why citrate and EDTA tubes were unacceptable.
Interference
studies showed that hemoglobin concentrations (>4 g/L) reduced the recovery of cTnT in serum
by >10%. Although the mechanism was not studied, the
interference
may be related to the initial absorbance
at
Table 2. cTnT and CK-MB in AMI and non-AMIpatients.
Hours after
onset of
chest pain
0-3
3-6
6-9
9-12
12-18
18-24
24-36
36-48
48-72
72-96
>96
Non-AMI
904
cTnT, ug/L
Total CK, U/L
CK-MB, pg/I-
No. of
subjects
20
22
17
29
31
40
53
11
24
17
18
116
Mean
SD
0.48
0.54
6.1
4.4
9.2
6.5
5.9
14.7
12.4
4.2
3.0
1.03
0.83
7.3
8.5
11.3
9.0
14.1
41.2
24.3
7.7
1.5
1.2
2.7
CLINICALCHEMISTRY,Vol.40, No.6, 1994
Range
0-4.8
0-3.5
0-25.4
0.04-42.1
0.17-45.5
0.05-33.7
0-98.2
0-146
0.04-95.8
0.12-34.2
0.87-5.78
0-16.0
Mean
SD
194
425
1298
150
654
1113
664
1065
734
1208
895
1507
266
143
2753
739
1320
1026
880
713
789
429
330
887
Range
20-650
62-2905
136-4010
144-2728
<20-3834
<20-2740
<20-7765
100-3405
67-7219
102-1126
77-629
<20-20375
Mean
SD
Range
6.5
26.3
97.0
64.4
114
73.1
64.5
57.3
40.2
22.7
14.5
8.8
7.8
41.4
102.1
66.2
112
78.8
200
97.9
72.8
22.6
12.6
13.5
0-35.2
0-76.3
2.9-335
5-203
1.2-330
1-326
0.3-1456
8.4-361
1-243
1.1-75.8
0.4-39.9
0-92.2
100
A
80.O-
0.2
207
9
0.4 0.3
I
I
I
I
10080604020
0
Specificity
WV
Specificity
0.201
B
80-
2
a,
Cl)
4020-
I
100
80
I
60
I
40
I
20
0
Specificity
Specificity
100
80
.60
j40
Fig. 5. Receiver-operatIng characteristic curves
for cTnT (U) and CK-MB (#{149})
vs time after onset
of chest pain: (A), 2-6 h; (B), 6-12 h; (C),
12-24 h; (0), 24-48 h; (E), 48-96 h.
Numbersindicate the cutoff concentrations for cTnT
and CK-M8 in paJL (at a Al of 2.5%). Circled values
are cutoffvaluesof 0.1 Mg/I and 5.0 pg/L used in the
current study for cTnT and CK-MB, respectively.
were added to the AM! group on the basis of increased
CK-MB (5). (When this minor injury group was included
in the non-AM!
group, the specificity
decreased
to 87%.)
Mair et al. reported a sensitivity of 96% from 96 patients
in the emergency department
(10). Patients in this population included those with unstable
angina, other cardiovascular
diseases, and miscellaneous
disorders.
We
believe that the discordance
in our data was due to differences in the inclusion criteria
for patients selected in
our study as compared
with others. We purposely
included
serum from critically ill patients and those with
multisystem
disorders.
We suggest that increased
cTnT
in some of these non-AM! patients was due to the exis-
20
0
100
80
60
40
20
0
Specificity
tence of minor myocardial
injuries not detected by CKMB.
Other explanations
for high cTnT in these patients
are possible. Patients with rhabdomyolysis
are likely to
have high smThT concentrations.
Any degree of crossreactivity
or nonspecific binding to smTnT in the cTnT
assay wifi produce serum concentrations
that exceed the
cutoff’. Previous studies have shown a 3.8% nonspecific
binding
to streptavidin-coated
tubes with the Boehringer
Mannheim
assay
of purified smTnT (16). However, this appears to be an artifact of the preparation
of
smTnT, because
there was no cross-reactivity
when
equivalent
smTnT concentrations
from serum samples
CUNICAL CHEMISTRY, Vol. 40, No. 6, 1994
905
0.4
3
0.2
-
-
CK-MB
Troponin-T
Fig. 8. Schematic comparison of CK-MBwithtroponin-Tfor detection
of minor myocardial injury: U, residual baseline content; El, new
onset, minor myocardlal injury; cutoff value.
-
0
0-3
3-6
6-9
9-12 122424-4848-7272-98
Hoursafteronset
Fig. 6. ClInical sensitivity of AMIfor cTnT (line) and CK-MB(bars) vs
time after onset of chest pain.
Cutoff values used were 0.1 pg/L for cTnT and 5.0
CK-MB.
Mg/L and
0
80
4..
(5
1
‘I-’
C
a,
C-,
60
C
0
C)
a, 40
>
4-.
(5
a,
I-
a,
20
0
0-3 I 6-9
3-6
9-12
12-24
24-48
48-96
Hours after onset
Fig. 7. Average concentrations of cTnT (#{149})
and CK-MB (#{149})
relative
to the upper limit of normal for 63 AMI patients studied.
The upper limit of normal
=
1.
were used (16). Also, cTnT may be upregulated
after
skeletal muscle injury. Although cTnT is absent in normal human skeletal muscles, cardiac and skeletal muscle forms are coexpressed
during fetal development
(24).
Fetal cTnT isoforms that are normally
absent in adult
cardiac tissue are transiently
expressed
in adult patients with failing hearts (25). Moreover,
fetal expression of cTnT has been demonstrated
in regenerating
skeletal muscle fibers of rats subjected to cold injury and
denervation
(26). However, there are no reports to date
to suggest that cardiac expression
of troponin
T occurs
after repair of skeletal muscle injury in humans.
The ROC curves (Fig. 5) show that cThT has a lower
clinical
specificity and diagnostic efficiency than CK-MB
in the diagnosis of AM!. If the objective of the cTnT assay
is diagnosis of AM!, our data suggest that CK-MB may
be superior to cThT during the first 48 h. However,
if
detection
of myocardial injury (to include AMI) is the
objective, then cThT maybe more sensitive
limit so that patients with minor myocardial injury may
have higher-than-normal
CK-MB
concentrations
that
are stifi below the cutoff. Some investigators
have lowered CK-MB limits to detect minor myocardial
injury (27,
28). However, the utility
of CK-MB will ultimately
be
limited by the inability
of the marker to distinguish myocardial injury from skeletal muscle damage. For cTnT,
selecting a cutoff at the upper limit of normal (Fig. 8)
could be useful for detecting
minor myocardial
injury
because there is no cTnT contribution
from skeletal muscles. A higher cTnT cutoff limit could be used to improve
clinical
specificity for AM! diagnosis;
however, the detection of minor myocardial injury will be lost.
The use of cTnT for detecting evidence of minor ischemia may have new applications
in the management
of
patients with unstable
angina.
Hamm eta!. showed that
patients with unstable
angina and abnormal cThT concentrations
were more likely to develop AMI or to die
during hospitalization (29). Collinson
et a!. reported
four patients with unstable angina
and abnormal cTnT
who developed cardiac events during the subsequent 6
months (23). Unstable angina patients identified as being at high risk may prompt cardiologists
to be more
aggressive
in treating their patients.
Our results for cTnT are consistent
with the concept
that irreversible myocardial
injury follows a progressive
and continuous pattern
of injury. This pattern
is characterized
by (a) minimal release of cytoplasmic proteins
after reversible injury; (b) development
of a subendocardial MI with a higher degree of protein release (small,
irreversible injury); and (c) progression
to a transmural
infarct with the highest degree of enzyme release (substantial
irreversible
injury).
Under this assumption,
many patients
with unstable angina will have injury
detectable
by increased
concentrations
of a sensitive
and specific marker such as cTnT.
The authors thank Boehringer Mannheim
for financial assistance and for supplying reagents and equipment needed to conduct
this study.
and specific
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CLINICALCHEMISTRY,Vol. 40, No. 6, 1994
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