Method for the in vitro diagnosis of the exclusion of
acute coronary syndromes
The present invention relates to a method for the in
vitro diagnosis of the exclusion of acute coronary
syndromes (ACS), commonly called myocardial infarction
(MI) , in patients presenting chest pain and suspected
of being at risk, according to which the quantification
of the concentration of at least one cardiac marker
selected from troponin, CK-MB and myoglobin and the
quantification of the concentration of D-dimers, based
on a sample, are associated.
Myocardial infarction is most commonly due to an abrupt
occlusive thrombosis of a coronary artery, resulting in
ischemic necrosis of a more or less extensive area of
the cardiac muscle. This acute coronary occlusion due
to a thrombus is most commonly associated with
fissuration or rupture of an atheroma plaque.
Myocardial infarction is a disease with a considerable
risk of fatality in the short or medium term and
constitutes an absolute cardiological emergency, the
incidence of which is still very high throughout the
world. Myocardial infarction most commonly manifests
itself at night or while resting, through intense pain
that appears abruptly in the chest, behind the sternum.
This pain resembles that of angina pectoris. In
practice, patients suffering from acute chest pain must
be transported as rapidly as possible to emergency
departments or EDs, in which, when the patient is
admitted, a series of examinations and tests is carried
out, including a study of the patient's clinical
history, an electrocardiogram and the assaying of
cardiac markers, such as troponin and/or CK-MB and/or
myoglobin. However, the assaying of cardiac enzymes,
CK-MB and myoglobin, has the drawback of not being
specific for the pathology, and practitioners have to
carry out other specialized examinations, such as
continuous electrocardiogram, or imaging techniques,
such as cardiac Doppler echography and coronography,
before putting forward a diagnosis. Such specialized
examinations are not available in all hospitals, and
rarely 24 hours a day. As regards the assaying of
troponin, since several hours may elapse during a
myocardial infarction before the troponin concentration
becomes elevated, and since the latter is only
detectable in the blood six hours after the beginning
of the chest pain, a further assaying of tropinin is
required four to six hours after the first troponin
assay in order to exclude a myocardial infarction in
patients who present a normal electrocardiogram, in
accordance with the NACB guidelines ["NACB Laboratory
Medicine Practice Guidelines: Characteristics and
Utilization of Biochemical Markers in ACS and Heart
Failure, CLINICAL: ACUTE CORONARY SYNDROMES - Chapter
1, 2004"].
There exists therefore today a real need to establish a
very early strategy for excluding patients suspected of
acute coronary syndrome when they are admitted to an
emergency department. A new strategy would make it
possible to limit the additional enzyme assays and/or
the supplementary examinations which represent an
additional cost, to avoid wasting time, to improve the
patient's comfort and possibly to direct said patient
to the appropriate department depending on his or her
pathology. In fact, the earlier the diagnosis, the
better the patient's care will be. In such an approach,
the negative prediction value and sensitivity are the
most important parameters.
In patent application US 6,309,888, it is proposed to
distinguish between the acute and nonacute stages of
coronary syndromes by searching for (1) a first marker,
such as malondialdehyde-modified LDLs, the presence of
which above a predetermined level indicates a very high
degree of diagnostic precision (i.e. the surface area
under the ROC or "Receiver Operating Characteristics"
curve is at least 0.875) for the presence of an acute
stage of ACS, then (2) either a second marker, such as
oxidized LDLs, the presence of which above a
predetermined level indicates a very high degree of
diagnostic precision for the presence of ACS, (2') or a
third marker, such as troponin I, the presence of which
above a predetermined level indicates a high degree of
diagnostic precision (i.e. the surface area under the
ROC or "Receiver Operating Characteristics" curve is at
least 0.70) for the presence of an acute stage of ACS,
(2") or the second and the third markers as defined
above.
Surprisingly, the applicant has found that associating
the quantification of the concentration of at least one
cardiac marker selected from troponin, CK-MB and
myoglobin and the quantification of the concentration
of D-dimers, based on a liquid sample from a patient
presenting chest pain, makes it possible to exclude the
diagnosis of an acute coronary syndrome with a high
sensitivity and a high negative prediction value, which
can reach up to approximately 100%, which enables both
a reduction in investigation costs and in time and an
acceleration of the approaches for shedding light on
the differential diagnosis.
A subject of the present invention is therefore a
method for the in vitro diagnosis of the exclusion of
an acute coronary syndrome based on a liquid sample
from a patient suspected of presenting a risk,
according to which:
the concentration of at least one cardiac marker
selected from troponin, CK-MB and myoglobin, in
said sample, is quantified,
the concentration of D-dimers in said sample is
quantified,
the concentration of said cardiac marker(s) of the
sample is compared to a predetermined threshold
concentration,
the concentration of D-diraers of the sample is
compared to a predetermined threshold
concentration of D-dimers, and
it is determined whether the concentration of said
cardiac marker(s) and the concentration of
D-dimers of the sample are respectively less than
the predetermined threshold concentration of said
cardiac marker(s) and than the predetermined
threshold concentration of D-dimers;
it being understood that the quantification of the
concentration of said cardiac marker(s) and the
quantification of the concentration of D-dimers are
carried out either sequentially and independently of
the order, or simultaneously, as explained in greater
detail below, based on the example of the
quantification of the concentration of troponin and the
quantification of the concentration of D-dimers.
According to the method of the invention, it is
therefore excluded that a patient is suffering from an
acute coronary syndrome when the concentrations of
cardiac marker (s) and of D-dimers are less than the
predetermined threshold concentrations.
In one embodiment of the invention,
the concentration of troponin in said sample is
quantified,
the concentration of D-dimers in said sample is
quantified,
the concentration of troponin of the sample is
compared to a predetermined threshold
concentration of troponin,
the concentration of D-dimers of the sample is
compared to a predetermined threshold
concentration of D-dimers, and
it is determined whether the concentration of
troponin and the concentration of D-dimers of the
sample are respectively less than the
predetermined threshold concentration of troponin
and than the predetermined threshold concentration
of D-dimers.
According to the method of the invention, it is
possible to exclude the diagnosis of an ACS, based on a
liquid sample from a patient presenting chest pain,
with a sensitivity and a negative prediction value that
are superior to the performance levels of the isolated
initial assaying of cardiac markers such as troponin
when the patient presents at the emergency department.
In fact, to date, although the performance level of the
new generations of troponin assays has improved, the
sensitivities and the negative prediction values do not
reach values in the region of 100%.
The quantification of the concentration of said cardiac
marker(s) and of the D-dimers can be carried out by any
method known to those skilled in the art for
determining a concentration of a marker in a liquid
sample. This quantification is preferably carried out
using at least one antibody specific for said cardiac
marker(s) or for the D-dimers, or at least one antibody
fragment specific for said cardiac marker(s).
Preferably, at least one monoclonal antibody is used,
for instance an anti-troponin monoclonal antibody. The
quantification of D-dimers is preferably carried out
using at least one anti-D-dimer antibody or at least
one anti-D-dimer antibody fragment. Preferably, at
least one anti-D-dimer monoclonal antibody is used.
The term "antibody fragment" is intended to mean in
particular P(ab)2, Fab, Fab' or sFv fragments [Blazar
et al., 1997, Journal of Immunology 159: 5821-5833 and
Bird et al., 1988, Science 242: 423-426] of a natural
antibody.
In the rest of the description which follows and in the
claims, the generic term "antibody" will be used to
denote, without distinction, a monoclonal antibody, a
monospecific polyclonal antibody or an antibody
fragment.
In one embodiment of the method of the invention, the
concentration of cardiac marker, preferably troponin,
in the sample is quantified and then the concentration
of D-dimers in the sample is quantified.
In a second embodiment of the method of the invention,
the concentration of D-dimers in the sample is
quantified and then the concentration of cardiac
marker, such as troponin, in the sample is quantified.
In a third embodiment of the method of the invention,
the concentration of cardiac marker, such as troponin,
and the concentration of D-dimers in the sample are
simultaneously quantified. In this embodiment, (i) at
least one anti-cardiac marker antibody (for example
anti-troponin) and at least one anti-D-dimer antibody,
corresponding to the definitions given above, can be
attached to one and the same solid phase, it being
possible for said solid phase to consist of a tube, a
strip or a cone (see, for example, the principle of the
VIDAS* HIV DUO test (reference 30 114) sold by the
applicant), or (ii) at least one anti-cardiac marker
antibody (for example, anti-troponin) is attached to
one solid phase and at least one anti-D-dimer antibody
is attached to another solid phase, said antibodies
corresponding to the definitions given above, and it
being possible for the solid phase to consist of a
tube, a strip, a cone or a particle.
A subject of the invention is also the use of a reagent
for quantifying the concentration of at least one
cardiac marker selected from troponin, CK-MB and
myoglobin, in a liquid sample, and of a reagent for
quantifying the concentration of D-dimers in a liquid
sample, said reagents being intended for use for the in
vitro diagnosis of the exclusion of an acute coronary
syndrome.
In one embodiment of the invention, said reagent for
quantifying the concentration of said cardiac marker(s)
is at least one antibody corresponding to the
definitions given above, and said reagent for
quantifying the concentration of D-dimers is at least
one antibody corresponding to the definitions given
above. Preferably, one of the reagents is an antitroponin
antibody and the other reagent is an anti-Ddimer
antibody.
The liquid sample is any liquid sample from the human
body that may contain the cardiac markers and the Ddimers.
Preferably, it is selected from serum, plasma
and blood.
Troponin is one of the protein components of striated
muscle. It is made up of three subunits, I, T and C,
which are involved in muscle contraction. Troponin I
and troponin T make it possible to differentiate
between a myocardial necrosis and a skeletal muscle
attack. One of the three isoforms of troponin, troponin
Ic, exhibits cardiac specificity with no cross
reaction. In the method of the invention, the
concentration of troponin T or the concentration of
troponin I, and preferably the concentration of
troponin Ic, is quantified.
Creatinin kinase, CK, is a dimer consisting of two
subunits denoted M and B, combined to give three
isoenzymes: CK-BB, CK-MB and CK-MM. These three
isoenzymes are located in the cytoplasm and each has a
molecular weight of approximately 82 000 daltons. The
CK in the serum of a normal adult man consists mainly
of the CK-MM isoenzyme with only trace amounts of CK-
MB. The CK-BB isoenzyme is not usually present in the
serum at the limits of detection in most CK assays. The
detection of significant amounts of CK-MB in the serum
is usually indicative of ACS. However, CK-MB has also
been found in the serum of patients exhibiting
disorders other than ACS.
Myoglobin is a low-molecular-weight protein derived
from the heme of muscle cells. It constitutes a marker
for irreversible cell necrosis, the serum level of
which increases early on after the beginning of
infarction. Its half-life, which is very brief, allows
it to closely reflect the evolution of the infarction.
In a population of normal individuals, the blood
myoglobin level is from 6 to 85 ng/ml. The diagnostic
threshold is set at between 70 and 90 ng/ml. In the
course of an MI, myoglobin appears between the 2nd and
the 4th hour, with a peak between the 9th and the 12th
hours. It remains assayable for from 7 to 20 hours
after the peak. The proportion of false negatives
depends on the amount of time between admission and the
beginning of chest pain and is from 45% to 67% before
the 4th hour. Myoglobin exists in all the striated
muscles of the organism. The false positives are
therefore the procedures carried out for cardiac
reanimation, external electric shocks, intramuscular
injections, and certain myopathies. The increase is
usually relatively insubstantial and the context often
makes it possible to identify them. The myoglobin level
also increases in the event of renal insufficiency and
in any situation which induces a decrease in glomerular
filtration, such as advanced cardiac insufficiency.
Age, weight and sex have little influence on blood
myoglobin levels. Similarly, blood myoglobin levels
usually remain normal after exercise.
D-dimers are products of fibrin degradation, soluble
fragments of very heterogeneous composition that result
from two simultaneous phenomena:
coagulation of fibrinogen to stabilized fibrin,
after the action of thrombin and of factor Xllla,
digestion of the fibrin clot, by plasrain, to
soluble fragments that are released into the
bloodstream. The end products of the lysis of the
clot are the D-dimers.
Among all the markers of thrombotic states, D-dimers
are the only ones that really attest to the presence of
stabilized fibrin.
Said cardiac marker(s), in particular troponin, and the
D-dimers can be quantified using various immunoassay
principles which are well known to those skilled in the
art, such as ELISA, ELFA, latex agglutination,
turbidimetry, turbidimetry with particles,
nephelometry, nephelometry with particles, or other
appropriate methods. These principles and other methods
are, for example, described in "Labor und Diagnose",
ed. L. Thomas, TH-Books Verlagsgesellschaft mbH,
Frankfurt, 1998, chapter 60 or in "Laboratory
Techniques in Biochemistry and Molecular Biology, - An
introduction to Radioimmunoassay and Related
Techniques" ed. T. Chard, Elsevier, Amsterdam, 1987.
Thus, the quantification of the concentration of said
cardiac marker(s), in particular of troponin, and the
quantification of the concentration of D-dimers in the
sample are carried out, independently of one another,
by means of an immunoassay, on the basis of an assay
selected from ELISA, ELFA, turbidimetry, nephelometry,
turbidimetry with particles, nephelometry with
particles, and latex agglutination.
The threshold concentration values can be defined by
those skilled in the art, individually for the cardiac
markers such as troponin, and the D-dimers, on the
basis of well-known procedures. By way of example, the
following procedure can be applied for the D-dimers:
the samples from a number of patients diagnosed as ACSpositive,
for example by imaging, and a number of
samples from ACS-negative individuals, are assayed for
the concentration of D-dimers; the results obtained are
evaluated on the basis of various threshold values and
the threshold concentration values that make it
possible to exclude the ACS-negatives and that do not
make it possible to exclude the ACS-positives are
selected. The threshold concentration values may vary
according to the technique and the kit used for the
D-dimer assay. This procedure is also applicable to the
cardiac markers.
In the specific case of troponin, the threshold is
determined in accordance with international
recommendations and must correspond to the 99th
percentile of a reference control population ["NACB
Laboratory Medicine Practice Guidelines:
Characteristics & Utilization of Biochemical Markers in
ACS and Heart Failure, CLINICAL: ACUTE CORONARY
SYNDROMES - Chapter 1, 2004"]. The threshold
concentration values may also vary according to the
technique and to the kit used for the troponin assay.
A preferred but nonlimiting embodiment of the present
invention is the method for the in vitro diagnosis of
the exclusion of an acute coronary syndrome in a
patient suspected of presenting a risk, as described
above, in which troponin, preferably troponin Ic, is
tested using a sandwich immunoenzymatic method on a
device such as the VIDAS instrument, by applying a
threshold concentration value of 0.1 ug/1.
Another preferred but nonlimiting embodiment of the
present invention is the method for the in vitro
diagnosis of the exclusion of an acute coronary
syndrome in a patient suspected of presenting a risk,
as described above, in which the D-dimers are tested
using a sandwich immunoenzymatic method on a device
such as the VIDAS" instrument, by applying a threshold
concentration value of 250 ng/ml.
The method for the in vitro diagnosis of the exclusion
of an ACS, described above, was validated by means of a
prospective study, carried out over six months, on 119
patients, from an emergency department, suffering from
chest pain. The method of the invention made it
possible to exclude the diagnosis of cardiac muscle
necrosis with a very high sensitivity and a very high
negative predictive value, superior to the performance
levels of assaying troponin alone at the time the
patient presented at the emergency department.
The method of the invention is described in greater
detail below and on the single figure representing the
algorithm used to evaluate the combined use of troponin
Ic and of D-dimers and to diagnose the patients
excluded from ACS. None of these details can be
considered to limit the invention in any way
whatsoever.
EXAMPLE
Materials and methods:
119 consecutive patients (88 men and 31 women)
presenting chest pain were selected in the emergency
department. The average age was 59 years old, more or
less thirteen years, in the study. An electrocardiogram
was carried out for all the patients. Fresh plasma
samples collected from these patients were tested with
the VIDAS' D-Dimer New kit (bioMerieux) and with the
VIDAS""' Troponin I (TNI) kit (bioMerieux) at different
times, on the VIDAS* instrument (bioMerieux).
In the study, the time TO corresponds to the appearance
of the chest pain, the time Tl corresponds to the time
at which the first sample was taken when the patient
arrived at the emergency department, and the time T2
corresponds to Tl plus 6 hours. A threshold value of
0.1 pg/1 was set for the troponin I, in accordance with
the new international recommendations (99th percentile
of a normal population) for excluding a myocardial
infarction. The optimal threshold value for the
D-dimers was determined for excluding myocardial
infarction. An ROC ("Receiver Operating
Characteristics") curve, for the D-dimers, for
predicting a myocardial infarction was established
using the Analyse-It program. The ROC curve for
excluding myocardial infarction for the D-dimers gives
an optimal threshold value at 250 ng/ml (Youden index:
14.3%, sensitivity: 88.5% and specificity: 25.8%; area
under the curve 0.60).
The algorithm used to evaluate the combined use of
troponin Ic and of D-dimers is described in the single
figure, in which DDi = D-dimers, Tnl = troponin I, MI =
myocardial infarction, UA = unstable angina, SA =
stable angina.
It can be seen on this figure that, if the
concentration of troponin Ic is greater than 0.1 ug/1
at time Tl arid if the concentration of D-dimers is less
than 250 ng/ml at time Tl, myocardial infarction cannot
be excluded. If the concentration of troponin Ic is
less than 0.1 ug/1 at time Tl and if the concentration
of D-dimers is less than 250 ng/ml at time Tl,
myocardial infarction is excluded, in accordance with
the method of the invention.
Sensitivity and apecificity:
(Table Removed)
Discussions
26 patients (22%) are diagnosed Mi-positive. Among
these patients, just one has a negative troponin and a
negative D-diraers at time TI. However, as for the other
25 patients who were troponin-negative and D-dimernegative
at Tl, the troponin of said patient remains
negative at T2. With the algorithm defined above, 21%
(25/119) of the population can be excluded in the
emergency departments in terms of an MI as soon as the
results of the assays for troponin and the D-dimers
carried out at time Tl are obtained.
A D-dimers-negative result (< 250 ng/ml) associated
with a troponin-negative result improves the
sensitivity of the diagnosis at time Tl, compared to
the result obtained with troponin alone, from 46.2% to
96.2%. With the combination of the two tests, the
sensitivity obtained at time Tl is equal to that
obtained with the troponin assay alone at time T2.
The association of the troponin and D-dimer assays is a
combination that allows a very early exclusion of
patients suspected of presenting a risk of MI in
emergency departments, with a very high sensitivity and
a very high negative prediction value.

WHAT IS CLAIMED IS
1. A method for the in vitro diagnosis of the
exclusion of an acute coronary syndrome based on a
liquid sample from a patient suspected of presenting a
risk, according to which:
the concentration of at least one cardiac marker
selected from troponin, CK-MB and myoglobin, in
said sample, is quantified,
the concentration of D-dimers in said sample is
quantified,
the concentration of said cardiac marker(s) of the
sample is compared to a predetermined threshold
concentration,
the concentration of D-dimers of the sample is
compared to a predetermined threshold
concentration of D-dimers, and
it is determined whether the concentration of said
cardiac marker(s) and the concentration of
D-dimers of the sample are respectively less than
the predetermined threshold concentration of said
cardiac marker(s) and than the predetermined
threshold concentration of D-dimers.
2. The method as claimed in claim 1, according to
which:
the concentration of troponin in said sample is
quantified,
the concentration of D-dimers in said sample is
quantified,
the concentration of troponin of the sample is
compared to a predetermined threshold
concentration of troponin,
the concentration of D-dimers of the sample is
compared to a predetermined threshold
concentration of D-dimers, and
it is determined whether the concentration of
troponin and the concentration of D-dimers of the
sample are respectively less than the
predetermined threshold concentration of troponin
and than the predetermined threshold concentration
of D-dimers.
3. The method as claimed in claim 2, according to
which:
the concentration of troponin in said sample is
quantified, then
the concentration of D-dimers in said sample is
quantified,
the concentration of troponin of the sample is
compared to a predetermined threshold
concentration of troponin,
the concentration of D-dimers of the sample is
compared to a predetermined threshold
concentration of D-dimers, and
it is determined whether the concentration of
troponin and the concentration of D-dimers of the
sample are respectively less than the
predetermined threshold concentration of troponin
and than the predetermined threshold concentration
of D-dimers,
4. The method as claimed in claim 2, according to
which:
the concentration of D-dimers in said sample is
quantified, then
the concentration of troponin in said sample is
quantified,
the concentration of troponin of the sample is
compared to a predetermined threshold
concentration of troponin,
the concentration of D-dimers of the sample is
compared to a predetermined threshold
concentration of D-dimers, and
it is determined whether the concentration of
troponin and the concentration of D-dimers of the
sample are respectively less than the
predetermined threshold concentration of troponin
and than the predetermined threshold concentration
of D-dimers.
5. The method as claimed in claim 2, according to
which:
the concentration of troponin and the
concentration of D-dimers in said sample are
simultaneously quantified,
the concentration of troponin of the sample is
compared to a predetermined threshold
concentration of troponin,
the concentration of D-dimers of the sample is
compared to a predetermined threshold
concentration of D-dimers, and
it is determined whether the concentration of
troponin and the concentration of D-dimers of the
sample are respectively less than the
predetermined threshold concentration of troponin
and than the predetermined threshold concentration
of D-dimers.
6. The method as claimed in claim 1 or claim 2, in
which the liquid sample is selected from serum, plasma
and blood.
7. The method as claimed in claim 1, in which the
quantification of the concentration of said cardiac
marker (s) arid the quantification of the concentration
of D-dimers in the sample are carried out,
independently of one another, by means of an
immunoassay.
8. The method as claimed in claim 2, in which the
quantification of the concentration of troponin and the
quantification of the concentration of D-dimers in the
sample are carried out, independently of one another,
by means of an immunoassay.
9. The method as claimed in claim 7, in which the
quantification of the concentration of said cardiac
marker(s) and the quantification of the concentration
of D-dimers in the sample are carried out,
independently of one another, on the basis of an assay
selected from ELISA, ELFA, turbidimetry, nephelometry,
turbidimetry with particles, nephelometry with
particles, and latex agglutination.
10. The method as claimed in claim 8, in which the
quantification of the concentration of troponin and the
quantification of the concentration of D-dimers in the
sample are carried out, independently of one another,
on the basis of an assay selected from ELISA, ELFA,
turbidimetry, nephelometry, turbidimetry with
particles, nephelometry with particles, and latex
agglutination.
11. The use of a reagent for quantifying the
concentration of at least one cardiac marker selected
from troponin, CK-MB and myoglobin, in a liquid sample,
and of a reagent for quantifying the concentration of
D-dimers in a liquid sample, intended for use for the
in vitro diagnosis of the exclusion of an acute
coronary syndrome.
12. The use, as claimed in claim 11, of a reagent for
quantifying the concentration of troponin in a liquid
sample and of a reagent for quantifying the
concentration of D-dimers in a liquid sample, intended
for use for the in vitro diagnosis of the exclusion of
an acute coronary syndrome.
13. The use as claimed in claim 11, in which one of
the reagents is at least one antibody for at least one
cardiac marker and the other of the reagents is at
least one anti-D-dimer antibody.
14. The use as claimed in claim 12, in which one of
the reagents is at: least one anti-troponin antibody and
the other of the reagents is at least one anti-D-dimer
antibody.
15. The use as claimed in any one of claims 11 to 14,
in which the liquid sample is selected from serum,
plasma and blood.