U.S. patent application number 15/472264 was filed with the patent office on 2017-09-14 for nt-pro anp and sflt-1 for the differentiation between circulatory and ischemic events.
The applicant listed for this patent is ROCHE DIAGNOSTICS OPERATIONS, INC. Invention is credited to Georg HESS, Andrea HORSCH, Dietmar ZDUNEK.
Application Number | 20170261517 15/472264 |
Document ID | / |
Family ID | 41402332 |
Filed Date | 2017-09-14 |
United States Patent
Application |
20170261517 |
Kind Code |
A1 |
HESS; Georg ; et
al. |
September 14, 2017 |
NT-pro ANP and SFlt-1 For The Differentiation Between Circulatory
And Ischemic Events
Abstract
The present disclosure relates to the field of laboratory
diagnostics. The present disclosure provides means and methods for
differentiating between an acute circulatory event and an ischemic
event, as the cause underlying an acute medical event of a
patient.
Inventors: |
HESS; Georg; (Mainz, DE)
; ZDUNEK; Dietmar; (Mannheim, DE) ; HORSCH;
Andrea; (Tutzing, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ROCHE DIAGNOSTICS OPERATIONS, INC |
Indianapolois |
IN |
US |
|
|
Family ID: |
41402332 |
Appl. No.: |
15/472264 |
Filed: |
March 28, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13461833 |
May 2, 2012 |
9638700 |
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15472264 |
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PCT/EP2010/066660 |
Nov 2, 2010 |
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13461833 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/6893 20130101;
G01N 2800/32 20130101; G01N 2800/347 20130101; G01N 2333/58
20130101; G01N 2333/71 20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 3, 2009 |
EP |
09174873.1 |
Claims
1: A method for rapidly differentiating whether an acute medical
event in an emergency patient is associated with (i) a circulatory
complication; (ii) an ischemic complication; or (iii) both a
circulatory complication and an ischemic complication; the method
comprising the steps of a) determining the amount of an ANP-type
peptide in a serum or plasma sample from a subject suffering from
an acute medical event; b) determining the amount of sFlt-1 peptide
in a serum or plasma sample from said subject suffering from an
acute medical event; c) comparing the amounts of the ANP-type
peptide and the sFlt-1 peptide measured in steps a) and b) to
reference amounts for ANP-type peptide and sFlt-1 peptide,
respectively; and d) establishing a differentiation based on the
results of c) by: (i) diagnosing an acute medical event associated
with a circulatory complication if an increased level of the
ANP-type peptide relative to the reference amount is determined,
(ii) diagnosing an acute medical event associated with an ischemic
complication if an increased level of sFlt-1 relative to the
reference amount is determined, and (iii) diagnosing an acute
medical event associated with both a circulatory complication and
an ischemic complication if an increased level of the ANP-type
peptide relative to the reference amount and an increased level of
sFlt-1 relative to the reference amount is determined.
2: The method of claim 1, wherein the ischemic complication
comprises systolic blood pressure of less than 80 mmHg.
3: The method of claim 1, wherein the acute medical event is heart
failure, lung failure or renal failure.
4: The method of claim 1, wherein the ischemic complication is an
acute hypoxia of the spleen, the heart, the kidney, the bowel or
the limbs.
5: The method of claim 1, wherein the ANP-type peptide is
NT-proANP.
6: The method of claim 5, wherein the reference amount for
NT-proANP is about 2500 pg/ml.
7: The method of claim 1, wherein the reference amount for sFlt-1
peptide is about 500 pg/ml.
8: The method of claim 1, further comprising the step of obtaining
a serum or plasma sample from a subject suffering from an acute
medical event before step a).
9: The method of claim 1, wherein the determining step a) is
performed on a portion of a serum or plasma sample from said
subject suffering from an acute medical event, and wherein step b)
is performed on a further portion of the same serum or plasma
sample.
10: The method of claim 1, wherein step a) comprises contacting, in
vitro, said serum or plasma sample from said subject with an
antibody immunoreactive for an NT-proANP peptide.
11: The method of claim 10, wherein step a) requires contacting
said sample with two antibodies immunoreactive for different
portions of the NT-proANP peptide, wherein the first antibody is a
polyclonal sheep NT-proANP-specific antibody and the second
antibody is conjugated with horseradish peroxidase (HRP).
12: The method of claim 10, wherein the antibody in step a) is
bound to a microtiterstrip.
13: The method of claim 1, wherein step b) comprises contacting, in
vitro, the serum or plasma sample with two antibodies
immunoreactive for different portions of a sFlt-1 peptide, wherein
the first antibody has one of a biotin and a streptavidin linked
thereto and wherein the second antibody has a Tris (2,2'-bipyridyl)
ruthenium (H)-complex linked thereto.
14: A device for rapidly differentiating whether an acute medical
event in an emergency patient is associated with (i) a circulatory
complication; (ii) an ischemic complication, or (iii) both a
circulatory complication and an ischemic complication; said device
comprising a) an analyzing unit for determining the amount of an
ANP-type peptide in a serum or plasma sample of said emergency
patient; b) an analyzing unit for determining the amount of sFlt-1
in a serum or plasma sample from said emergency patient; c) a
computer unit for comparing the measured amounts of the ANP-type
peptide and sFlt-1 to reference amounts for ANP-type peptide and
sFlt-1 peptide, respectively; and a computer unit for establishing
a diagnosis based on the results of the comparison, wherein (i) an
acute medical event associated with a circulatory complication is
diagnosed if an increased level of the ANP-type peptide relative to
the reference amount is determined, (ii) an acute medical event
associated with an ischemic complication is diagnosed if an
increased level of sFlt-1 relative to the reference amount is
determined, and (iii) an acute medical event associated with both a
circulatory complication and an ischemic complication is diagnosed
if an increased level of the ANP-type peptide relative to the
reference amount and an increased level of sFlt-1 relative to the
reference amount is determined.
15: The device of claim 14, wherein the ANP-type peptide is proANP.
Description
PRIORITY CLAIM
[0001] This application is a continuation of International
Application No. PCT/EP2010/066660, filed Nov. 2, 2010, which claims
the benefit of European Patent Application No. 09174873.1, filed
Nov. 3, 2009, the disclosures of which are hereby incorporated by
reference in their entirety.
FIELD OF THE DISCLOSURE
[0002] The present disclosure relates to the field of medical
diagnostics. More specifically, the present disclosure relates to
diagnostic applications, and systems for performing the same, which
may be used in the diagnosis of one or more causes underlying an
acute medical event of a patient.
BACKGROUND OF THE DISCLOSURE
[0003] Patients in emergency units or emergency departments of
hospitals often present with suddenly developing, potentially life
threatening conditions. The extent and the duration of said
conditions are frequently unknown. The first steps of therapy are
generally, and necessarily, directed at the support of the
patient's vital functions such as mechanical ventilation, fluid
supply, blood transfusion, defibrillation, external pacing or
pharmacotherapy (for example, the administering epinephrine or
vasopressin for low blood pressure; administering amiodarone,
lidocaine, procainamide or magnesia sulphate for ventricular
fibrillation; administering epinephrine, atropine or sodium
bicarbonate for asystole). However, in most cases the impaired
vital functions are only the symptom of another pre-existing
condition. A successful therapy of the patient, thus, requires
treatment of the cause underlying the acute condition. In cases of
trauma the cause may be obvious. In other cases it may be more
difficult to find.
[0004] In many cases, the underlying causes of a patient's symptoms
(which may be life-threatening) are not readily apparent. For
example, dyspnea can be caused by such diverse conditions as heart
failure, pneumonia, sepsis, acute respiratory distress syndrome and
pulmonary embolism. Syncope, the transient loss of consciousness
and postural tone, may occur suddenly without warning or may be
preceded by symptoms such as lightheadedness, dizziness, a feeling
of warmth, nausea, diaphoresis and visual blurring. The ability of
medical professionals to differentiate between syncope and seizure,
for example, is important and in some cases difficult.
[0005] A pathophysiological mechanism underlying syncope is vasal
dysregulation. Vasal dysregulation can have a variety of causes.
For example, cardial causes include pulmonary embolism, acute
myocardial infarction, cardiac arrhythmias (bradyarrhythmias as
well as tachyarrhythmias) or hypertrophic obstructive
cardiomyopathy. Syncope can also be caused by the activation of the
parasympathetic nervous system which may be triggered by events
such as painful or unpleasant stimuli, prolonged standing, rapid
change from a reclining into an upright position, hyperthermia or
urination. Another unspecific symptom that may be associated with
life threatening conditions is acute chest discomfort. This symptom
can be caused by stable angina, acute cardiovascular events
(unstable angina or myocardial infarction), pulmonary embolism,
peptic ulcer or pneumonia, for example.
[0006] Differential diagnosis takes into account the history of the
patient and includes a clinical examination. These procedures may
be time consuming and the administration of analgetics may reduce
the diagnostic value of a clinical examination. Moreover, it is
difficult or impossible to get information on history of the
patient if the patient suffers from reduced consciousness or is
mechanically ventilated. However, a rapid diagnosis allows a rapid
initiation of a suitable therapy, reduces the suffering of the
patient and increases his/her chances of survival.
[0007] Clinically, ischemic events (such as those described above)
are characterized by pain, paleness of the skin, and weak or absent
pulse in the affected area. Additionally, imaging methods may be
used for the characterization of ischemic events, such as
ultrasonography, computed tomography, magnetic resonance imaging
(with and without contrast agent), angiography and scintigraphy may
be used.
[0008] Circulatory complications may be detected by the presence of
abnormal blood pressure. Occasionally, the presence of cardiac
arrhythmia and acute cardiovascular events may be used as an
additional indicator for circulatory complications. However, these
and other known methods for detecting circulatory events do not
allow for the diagnosis of temporary complications. Moreover, the
methods known in the art do not yield quantitative information
about the severity of a circulatory complication
[0009] Symptoms and complications associated with ischemic events,
for example, may be diagnosed with an initial evaluation of
chest-pain patients including an electrocardiogram (ECG) and
cardiac markers such as troponins. These tests, while specific, may
be insensitive and can leave the requirement for further testing to
achieve an accurate diagnosis. Other methods of diagnosis include
magnetocardiography imaging which utilizes superconducting quantum
interference devices to detect the weak magnetic fields generated
by the heart's electrical fields (which utilizes the correlation
between abnormal cardiac depolarisation or repolarisation and
abnormality in the magnetic field map). Magnetocardiography imaging
is approved by the Food and Drug Administration (FDA) as a safe
device for the non-invasive detection of ischemia. However, in
general these methods are generally, complicated, slow and not very
sensitive.
SUMMARY OF THE DISCLOSURE
[0010] The present disclosure provides means and methods to
differentiate between an acute circulatory event and an ischemic
event as the cause underlying an acute medical event of a
patient.
[0011] According an embodiment of the disclosure, a method for
rapidly diagnosing if an acute medical event in an emergency
patient is associated with a circulatory and/or an ischemic
complication is provided. The method comprises the steps of: a)
determining the amount of an ANP-type peptide in a sample of a
patient; b) determining the amount of sFlt-1 in a sample from a
patient; c) comparing the amounts measured in steps a) and b) to
reference amounts; and establishing a diagnosis based on the
results of c), and d) establishing a diagnosis based on the results
of c), wherein an increased level of the ANP-type peptide relative
to the reference amount is indicative of a circulatory complication
and wherein an increased level of sFlt-1 relative to the reference
amount is indicative of an ischemic complication, wherein the
circulatory complication is caused by cardiac arrhythmia, and
wherein the ischemic complication is characterized by a systolic
blood pressure of less than 80 mmHg.
[0012] According to some embodiments, increased levels of sFlt-1
and the ANP-type peptide relative to the reference amounts are
indicative of a combined circulatory and ischemic complication.
[0013] In some embodiments, a method for diagnosing one of a
circulatory complication and an ischemic complication associated
with an acute medical event in a subject is provided. The method
includes the steps of contacting, in vitro, a portion of a sample
from a subject with an antibody immunoreactive for an ANP-type
peptide; contacting, in vitro, a portion of the sample from the
subject with an antibody immunoreactive for a sFlt-1 peptide;
determining the amounts of the ANP-type peptide and the sFlt-1
peptide in the sample based on said steps of contacting. The method
also includes the steps of comparing the amounts of the ANP-type
peptide and the sFlt-1 peptide (determined in the steps of
determining) with reference amounts for ANP-type peptide and sFlt-1
peptide, respectively. Such methods also include the steps of
diagnosing one of a circulatory complication if the amount of
ANP-type peptide determined in said step of determining is greater
than the reference amount for ANP-type peptide and an ischemic
complication if the amount of sFlt-1 peptide determined in said
step of determining is greater than the reference amount for sFlt-1
peptide.
[0014] In some embodiments, the ANP-type peptide is NT-proANP or a
variant thereof. In some such embodiments, the reference amount for
NT-proANP is about 2500 pg/ml.
[0015] According to some embodiments, the reference amount for
sFlt-1 is about 500 pg/ml.
[0016] According to other embodiments of the disclosure, a system
for diagnosing one of a circulatory complication or an ischemic
complication associated with an acute medical event in a subject is
provided. According to some such embodiments, the system includes
an analyzing unit comprising means for contacting, in vitro, a
portion of a sample from a subject with an antibody immunoreactive
for an ANP-type peptide and means for determining the amount of the
ANP-type peptide in the sample and an analyzing unit comprising
means for contacting, in vitro, a portion of the sample from the
subject with an antibody immunoreactive for an sFlt-1 peptide and
means for determining the amount of the sFlt-1 peptide in the
sample. The system also includes a computing device having a
processor and a non-transient machine readable media including a
plurality of instructions executable by the processor, the
instructions, when executed compare the amount of ANP-type peptide
determined by the analysing unit comprising means for determining
the amount of ANP-type peptide to a reference amount of ANP-type
peptide, compare the amount of sFlt-1 peptide determined by the
analysing unit comprising means for determining the amount of
sFlt-1 peptide to a reference amount of sFlt-1 peptide, provide a
diagnosis of a circulatory complication if the amount of ANP-type
peptide in the sample is greater than the reference amount for
ANP-type peptide, and provide a diagnosis of an ischemic
complication if the amount of sFlt-1 peptide in the sample is
greater than the reference amount for sFlt-1 peptide.
[0017] In yet other embodiments of the instant disclosure, a kit
for facilitating a diagnosis of one of a circulatory complication
and an ischemic complication associated with an acute medical event
in a subject is provided. Some embodiments of such a kit include a
first antibody with specific binding affinity to an ANP-type
peptide or a variant thereof, a first antibody with specific
binding affinity to a sFlt-1 peptide or a variant thereof, a second
antibody with specific binding affinity to a portion of the
ANP-type peptide or a variant thereof different than the first
antibody with specific binding affinity to the ANP-type peptide or
variant thereof, the second antibody having a reporting molecule
linked thereto, and a second antibody with specific binding
affinity to a portion of the sFlt-1 peptide or a variant thereof
different than the first antibody with specific binding affinity to
the sFlt-1 peptide or variant thereof, the second antibody having a
reporting molecule linked thereto. Additionally, in some
embodiments of a kit according to the instant disclosure,
literature is provided. The literature may include reference amount
for ANP-type peptide, a reference amount for sFlt-1 peptide, a
diagnosis of a circulatory complication if a measured amount of
ANP-type peptide in a sample of the subject, determined using the
first and second antibody with specific binding affinity to the
ANP-type peptide, is greater than the reference amount for ANP-type
peptide, and a diagnosis of an ischemic complication if the amount
of sFlt-1 peptide in the sample of the subject, determined using
the first and second antibody with specific binding affinity to the
sFlt-1 peptide, is greater than the reference amount for sFlt-1
peptide.
[0018] According to some embodiments of the kits disclosed herein,
the first antibody with specific binding affinity to the ANP-type
peptide is immobilized.
[0019] In yet some further embodiments of kits disclosed herein, a
first reference standard having the reference amount for ANP-type
peptide; and a second reference standard having the reference
amount for sFlt-1 peptide are also provided.
DETAILED DESCRIPTION OF THE EMBODIMENTS OF THE DISCLOSURE
[0020] The embodiments disclosed herein are not intended to be
exhaustive or limit the disclosure to the precise form disclosed in
the following detailed description. Rather, the embodiments are
chosen and described so that others skilled in the art may utilize
their teachings.
[0021] According to the present disclosure, a system and method for
a rapid diagnostic test which can be used at the point of care to
aid diagnosis of the causes underlying critically reduced vital
functions in a patient is provided. The system and methods provided
herein surprisingly and unexpectedly provide for diagnosing if an
acute medical event in an emergency patient is associated with a
circulatory and/or an ischemic complication. The instant disclosure
also provides devices and kits for utilizing the system and methods
disclosed herein.
[0022] According to some embodiments of the present disclosure, a
method for rapidly diagnosing if an acute medical event in an
emergency patient is associated with a circulatory and/or an
ischemic complication is provided. In some embodiments, the method
includes the steps of: [0023] a) determining the amount of an
ANP-type peptide in a sample of a patient; [0024] b) determining
the amount of sFlt-1 in a sample from a patient; [0025] c)
comparing the amounts measured in steps a) and b) to reference
amounts; and [0026] d) establishing a diagnosis based on the
results of c).
[0027] The term "acute medical event" refers to a condition of a
patient which induces or causes the patient to seek medical
assistance. For example, the condition may be a serious,
potentially life threatening condition such as a failure of one or
more vital body functions or a relatively non-serious condition. A
failure of one or more vital body functions may refer to the sudden
failure of organs whose functions (or function) are essential for
survival. Also the term may be used to refer to the sudden
deterioration of a previously stable condition. Exemplary organs
whose functions are essential for survival include the lung, the
heart, (at least one of) the kidneys and the liver. Consequences of
organ failure depend on the organ in question. Signs of organ
failure depend on the affected organ and may include, pain,
metabolic acidosis, anuria, hepatic encephalopathy, insufficient
oxygenation of the blood and loss of consciousness. However, the
diagnosis of organ failure on its own does not give enough
indication of a suitable therapy.
[0028] The term "circulatory complication," as used herein, may
refer to a sudden deterioration of the function of the heart. Such
deterioration may be caused by cardiac arrhythmia, transient
cardiac arrest or pulmonary embolism, for example. Cardiac
arrhythmia can occur in two forms: bradyarrhythmia and
tachyarrhythmia. In bradyarrhythmia the frequency of heartbeat is
pathologically decreased in comparison to a healthy subject, for
example in bradyarrhythmia the heart rate is likely lower than
about 60 beats per minute. The most frequent forms of
bradyarrhythmia include sinus bradycardia, sinoatrial block, sinus
arrest, sick sinus syndrome and atriventricular block. In
tachyarrhythmia the frequency is pathologically increased when
compared with a healthy subject, for example in tachyarrhythmia the
heart rate is likely higher than about 100 beats per minute. Most
cases of tachyarrhythmia include at least one of supraventricular
tachycardia (associated with structural cardiovascular disease),
atrial fibrillation (associated with Wolff-Parkinson-White
syndrome), atrial flutter (associated with 1:1 atrioventricular
conduction), and ventricular tachycardia. Pulmonary embolism may be
caused by the occlusion of a pulmonary artery by a blood clot
(thromboembolism) or an air bubble (air embolism). In some
instances, blood clots may be formed in the pelvic or lower
extremity veins and migrate to the pulmonary arteries where they
get occlude an artery (or arteriole or the like). An air embolism
may be caused by a diving accident or by leaky venous catheters,
for example. Symptoms of pulmonary embolism include chest pain,
dyspnea and hemoptysis (coughing of blood). The pressure in the
lung circulation may rise and may cause right ventricular
failure.
[0029] The term "ischemic complication," as used herein may, refer
to a suddenly occurring hypoxia in any tissue or organ. For
example, the term may refer to ischemia of the spleen, bowel,
kidney, heart or one or more limbs. Acute ischemia may be caused by
the formation of blood clots in an artery of the systemic
circulation. The parts of the organ that rely on the occluded
artery for their blood supply may then be (at least partially) cut
off from their needed blood supply. Decreased blood pressure is
another frequent cause of ischemia. Depending on the duration of
ischemia, the oxygen demand of the ischemic tissue and the
remaining blood supply, the affected tissue may begin to die by
necrosis (and eventually die by necrosis). An ischemic complication
as referred to herein may be characterized by a systolic blood
pressure of less than 80 mmHg. Moreover, the ischemic complication
may also be characterized by organ specific pain, a reduced pulse
in the affected area and/or paleness of the skin.
[0030] An acute medical event, as used herein, may be associated
with circulatory and/or ischemic complications if such
complications precede or accompany the acute medical event, for
example. If such complications precede the acute medical event they
may take place 1, 2, 3 or 4 hours before the patient seeks medical
assistance. Thus, in some instances, the acute medical event may
actually be caused by (or enhanced by) such complications.
[0031] The term "diagnosing if an acute medical event in an
emergency patient is associated with a circulatory and/or an
ischemic complication" as used herein refers to identifying the
pathophysiological disorder or condition accompanying or preceding
the acute medical event from which the patient is suffering or
which was diagnosed when the patient presented in the hospital or
emergency unit. As will be understood by those skilled in the art,
such an assessment is usually not intended to be correct for 100%
of the subjects to be diagnosed. The term, however, intends that a
statistically significant portion of subjects can be correctly
diagnosed to suffer from the disease or condition. Whether a
portion is statistically significant can be determined by a person
skilled in the art using various well known statistic evaluation
tools including, for example determination of confidence intervals,
p-value determination, Student's t-test, Mann-Whitney test, etc.
Details may be found in Dowdy and Wearden, Statistics for Research,
John Wiley & Sons, New York 1983. According to the instant
disclosure, exemplary confidence intervals include at least
approximately 90%, at least approximately 95%, at least
approximately 97%, at least approximately 98% or at least
approximately 99%, for example. The p-values, according to the
instant disclosure, may include approximately 0.1, 0.05, 0.01,
0.005, or 0.0001, for example. Additionally, the probability
envisaged by the present disclosure allows that a diagnosis will be
correct for at least approximately 60%, at least approximately 70%,
at least approximately 80%, or at least approximately 90% of the
subjects of a given cohort or population.
[0032] According to some embodiments of the present disclosure, a
diagnosis that the patient has suffered an ischemic and/or
circulatory complication is indicative of the patient suffering
from a severe, potentially life threatening condition and requires
close medical attention.
[0033] An assessment according to methods disclosed herein,
includes a rapid assessment. A rapid assessment may be performed at
the point of care. Results of an assessment may be available in
less than about 120 minutes, and in some cases even less than about
60 or even about 30 minutes after admission of the patient to the
emergency unit (or admission to the emergency ambulance or after
the first physician or medical personal was consulted, for
example). In some instances, an assessment may be available in less
than about 60 minutes after the patient first seeks medical
assistance, for example.
[0034] According to some embodiments of the instant disclosure,
methods disclosed herein may be practised in an emergency unit. As
used herein, the term "emergency unit" may refer to any location
where individuals/patients with a (real or suspected) medical
emergency condition consult a person having a medical background,
for example a physician, to have an analysis of their pathological
state and the cause underlying their condition. Typical examples
include emergency departments or emergency rooms in hospitals,
emergency ambulances, doctor's offices and other institutions
suitable for treatment of patients.
[0035] An "emergency patient" may be a patient presenting to an
emergency unit or a patient who, from the perspective of a
physician--based on the patient's symptoms and medical history,
should present to an emergency unit.
[0036] According to the instant disclosure, some embodiments of the
methods disclosed herein include in vitro methods. For example, the
determination of the respective peptide (an ANP-type peptide or
sFlt-1) or peptides to be determined may be carried out in
vitro.
[0037] Additionally, methods disclosed herein may be used in
combination with other diagnostic methods. A person of skill in the
art will understand that a differential diagnosis may require a
combination of the patient's history, a clinical examination, and
laboratory tests. Further, according to some embodiments, the
methods of the present disclosure may be applied to guide the
further examination of a patient and to exclude unnecessary
tests.
[0038] In some embodiments of the instant disclosure, further
examination of the patient may utilize imaging methods.
Ultrasonography, computed tomography, magnetic resonance imaging
(with and without contrast agent), angiography and scintigraphy are
some such exemplary imaging methods.
[0039] The term "ANP-type peptide" refers to ANP, proANP,
pre-proANP and NT-proANP. ANP is synthesized and secreted by the
atria. Mature ANP is generated by sequential cleavage of pre-proANP
comprising 151 amino acids. Cleavage of a signal peptide (25 amino
acids) gives proANP (126 amino acids). Upon secretion the
propeptide is split into the biologically active ANP (28 amino
acids and the inactive N-terminal moiety (98 amino acids). The
turnover of ANP is rapid as its half-life is only 2.5 minutes in
blood. ANP promotes systemic arterial dilation, natriuresis,
diuresis and renin inhibition, blood pressure is, thus, decreased
by the action of ANP (see for example, Bonow, 1996, Circulation 93:
1946-1950).
[0040] The term "soluble Flt-1" or "sFlt-1" as used herein refers
to a polypeptide which comprises a soluble form of the VEGF
receptor Flt1. It was identified in conditioned culture medium of
human umbilical vein endothelial cells. The endogenous soluble Flt1
(sFlt1) receptor is chromatographically and immunologically similar
to recombinant human sFlt1 and binds [125I] VEGF with a comparable
high affinity. Human sFlt1 is shown to form a VEGF-stabilized
complex with the extracellular domain of KDR/Flk-1 in vitro. sFlt1
may refer to human sFlt1, for example, such as human sFlt1 deduced
from the amino acid sequence of Flt-1 as shown in Genebank
accession number P17948, GI: 125361. An amino acid sequence for
mouse sFlt1 is shown in Genebank accession number BAA24499.1, GI:
2809071.
[0041] The terms "ANP-type peptide" and "sFlt-1" used herein also
encompass variants of the aforementioned ANP-type or sFlt-1
polypeptides. Such variants have at least the same essential
biological and immunological properties as the specific ANP-type or
sFlt-1 polypeptides. In particular, they share the same essential
biological and immunological properties, for example, if they are
detectable by the same specific assays referred to in this
specification, e.g., by ELISA assays using polyclonal or monoclonal
antibodies specifically recognizing the said ANP-type or sFlt-1
polypeptides. Moreover, it is to be understood that a variant as
referred to in accordance with the present disclosure shall have an
amino acid sequence which differs due to at least one amino acid
substitution, deletion and/or addition wherein the amino acid
sequence of the variant is still at least 50%, 60%, 70%, 80%, 85%,
90%, 92%, 95%, 97%, 98%, or 99% identical with the amino sequence
of the specific ANP-type or sFlt-1 polypeptide, over the entire
length of the specific ANP-type peptide (e.g. human ANP, human
proANP, human NTproANP) or of the specific human sFlt-1,
respectively. The degree of identity between two amino acid
sequences can be determined by algorithms well known in the art.
The degree of identity may be determined by comparing two optimally
aligned sequences over a comparison window, where the fragment of
amino acid sequence in the comparison window may comprise additions
or deletions (e.g., gaps or overhangs) as compared to the reference
sequence (which does not comprise additions or deletions) for
optimal alignment. The percentage may be calculated by determining
the number of positions at which the identical amino acid residue
occurs in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the window of comparison and multiplying the result by
100 to yield the percentage of sequence identity. Optimal alignment
of sequences for comparison may be conducted by the local homology
algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981), by
the homology alignment algorithm of Needleman and Wunsch J. Mol.
Biol. 48:443 (1970), by the search for similarity method of Pearson
and Lipman Proc. Natl. Acad Sci. (USA) 85: 2444 (1988), by
computerized implementations of these algorithms (GAP, BESTFIT,
BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software
Package, Genetics Computer Group (GCG), 575 Science Dr., Madison,
Wis.), or by visual inspection. Given that two sequences have been
identified for comparison, GAP and BESTFIT may be employed to
determine their optimal alignment and, thus, the degree of
identity. Preferably, the default values of 5.00 for gap weight and
0.30 for gap weight length may be used. Variants referred to above
may be allelic variants or any other species specific homologs,
paralogs, or orthologs. Moreover, the variants referred to herein
include fragments or subunits of the specific ANP-type or sFlt-1
polypeptides or the aforementioned types of variants as long as
these fragments have the essential immunological and biological
properties as referred to above. Such fragments may be, e.g.,
degradation products of the ANP-type or sFlt-1 peptides. Further
included are variants which differ due to posttranslational
modifications such as phosphorylation or myristylation.
[0042] Determining the amount of an ANP-type peptide or sFlt-1 or
any other peptide or polypeptide referred to in this specification
relates to measuring the amount or concentration, for example as
semi-quantitatively or quantitatively. Measuring can be done
directly or indirectly. Direct measuring, in general, relates to
measuring the amount or concentration of the peptide or polypeptide
based on a signal which is obtained from the peptide or polypeptide
itself and the intensity of which directly correlates with the
number of molecules of the peptide present in the sample. Such a
signal--sometimes referred to herein as an intensity signal--may be
obtained, by measuring an intensity value of a specific physical or
chemical property of the peptide or polypeptide. Indirect measuring
includes measuring of a signal obtained from a secondary component
(i.e. a component not being the peptide or polypeptide itself) or a
biological read out system, for example, including measurable
cellular responses, ligands, labels, or enzymatic reaction
products.
[0043] In accordance with the present invention, determining the
amount of a peptide or polypeptide can be achieved by any known
means for determining the amount of a peptide in a sample. Such
means may comprise immunoassay devices and methods which utilize
labelled molecules in various sandwich, competition, or other assay
formats. Such assays may develop a signal which is indicative of
the presence or absence of the peptide or polypeptide. Moreover,
the signal strength can be correlated directly or indirectly (e.g.,
reverse or proportional) to the amount of polypeptide present in a
sample. Further suitable methods comprise measuring a physical or
chemical property specific for the peptide or polypeptide such as
its precise molecular mass or NMR spectrum. Such methods may
comprise biosensors, optical devices coupled to immunoassays,
biochips, analytical devices such as mass-spectrometers,
NMR-analyzers, or chromatography devices. Further, methods may
include micro-plate ELISA-based methods, fully-automated or robotic
immunoassays (available for example on Elecsys.TM. analyzers), CBA
(an enzymatic Cobalt Binding Assay, available for example on
Roche-Hitachi.TM. analyzers), and latex agglutination assays
(available for example on Roche-Hitachi.TM. analyzers).
[0044] Determining the amount of a peptide or polypeptide may
comprise the steps of (a) contacting a cell capable of eliciting a
cellular response the intensity of which is indicative of the
amount of the peptide or polypeptide with the said peptide or
polypeptide for an adequate period of time, and (b) measuring the
cellular response. For measuring cellular responses, the sample or
processed sample may be added to a cell culture and an internal or
external cellular response may be measured. The cellular response
may include the measurable expression of a reporter gene or the
secretion of a substance such as a peptide, polypeptide, or a small
molecule. The expression or substance shall generate an intensity
signal which correlates to the amount of the peptide or
polypeptide.
[0045] According to the embodiments of the instant disclosure,
determining the amount of a peptide or polypeptide may comprise the
step of measuring a specific intensity signal obtainable from the
peptide or polypeptide in the sample. As described above, such a
signal may be the signal intensity observed at an m/z variable
specific for the peptide or polypeptide observed in mass spectra or
a NMR spectrum specific for the peptide or polypeptide.
[0046] Additionally, according to some embodiments, determining the
amount of a peptide or polypeptide may comprise the steps of (a)
contacting the peptide with a specific ligand, (b) (optionally)
removing non-bound ligand, and (c) measuring the amount of bound
ligand. In some such embodiments, the bound ligand generates an
intensity signal. Binding may include both covalent and
non-covalent binding. A ligand according to the present invention
can be any compound including, for example, a peptide, polypeptide,
nucleic acid, or small molecule, binding to the peptide or
polypeptide described herein. Exemplary ligands include antibodies,
nucleic acids, peptides or polypeptides such as receptors or
binding partners for the peptide or polypeptide and fragments
thereof comprising the binding domains for the peptides, and
aptamers, for example nucleic acid or peptide aptamers. Methods to
prepare such ligands may include any method as is known in the art.
For example, identification and production of suitable antibodies
or aptamers may also include services offered by commercial
suppliers. The person skilled in the art is familiar with methods
to develop derivatives of such ligands with higher affinity or
specificity. For example, random mutations can be introduced into
the nucleic acids, peptides or polypeptides. These derivatives can
then be tested for binding according to screening procedures known
in the art, e.g. phage display. Antibodies as referred to herein
include both polyclonal and monoclonal antibodies, as well as
fragments thereof, such as Fv, Fab and F(ab).sub.2 fragments that
are capable of binding antigen or hapten. The present invention
also includes single chain antibodies and humanized hybrid
antibodies wherein amino acid sequences of a non-human donor
antibody exhibiting a desired antigen-specificity are combined with
sequences of a human acceptor antibody. The donor sequences will
usually include at least the antigen-binding amino acid residues of
the donor but may comprise other structurally and/or functionally
relevant amino acid residues of the donor antibody as well. Such
hybrids may be prepared any method known in the art. According to
embodiments of the present disclosure, the ligand or agent binds
specifically to the peptide or polypeptide. Specific binding
according to the present invention means that the ligand or agent
should not bind substantially to ("cross-react" with) another
peptide, polypeptide or substance present in the sample to be
analyzed. According to various embodiments of the instant
disclosure, the specifically bound peptide or polypeptide should be
bound with at least about 3 times higher, at least about 10 times
higher or even at least about 50 times higher affinity than any
other relevant peptide or polypeptide. Non-specific binding may be
tolerable, if it can still be distinguished and measured
unequivocally, for example, according to its size on a Western
Blot, or by its relatively higher abundance in the sample. Binding
of the ligand can be measured by any method known in the art. In
some embodiments, the method disclosed herein is semi-quantitative
or quantitative. Exemplary methods are described in the
following.
[0047] First, according to embodiments of the instant disclosure,
binding of a ligand may be measured directly, for example, by NMR
or surface plasmon resonance.
[0048] Next, if the ligand serves as a substrate of an enzymatic
activity of the peptide or polypeptide of interest, an enzymatic
reaction product may be measured (e.g., the amount of a protease
can be measured by measuring the amount of cleaved substrate on a
Western Blot for example). According to some embodiments, the
ligand may exhibit enzymatic properties itself and the
"ligand/peptide or polypeptide" complex or the ligand which was
bound by the peptide or polypeptide, respectively, may be contacted
with a suitable substrate allowing detection by the generation of
an intensity signal. For measurements of enzymatic reaction
products, the amount of substrate may be saturating. The substrate
may also be labeled with a detectable label prior to the reaction.
In some embodiments, the sample may be contacted with the substrate
for an adequate period of time. As used herein, an adequate period
of time includes the time necessary for an detectable amount of
product to be produced. According to some embodiments, instead of
measuring the amount of product, the time necessary for appearance
of a given (detectable) amount of product can be measured.
[0049] Next, in some embodiments, the ligand may be coupled
covalently or non-covalently to a label allowing detection and
measurement of the ligand. Labelling may be done by direct or
indirect methods. Direct labeling involves coupling of the label
directly (covalently or non-covalently) to the ligand. Indirect
labeling involves binding (covalently or non-covalently) of a
secondary ligand to the first ligand. In some embodiments, the
secondary ligand specifically binds to the first ligand. The
secondary ligand may be coupled with a suitable label and/or be the
target (receptor) of tertiary ligand binding to the secondary
ligand. The use of secondary, tertiary or even higher order ligands
may be used to increase the signal. Exemplary secondary and higher
order ligands may include antibodies, secondary antibodies, and a
streptavidin-biotin system (for example, from Vector Laboratories,
Inc.). The ligand or substrate may also be "tagged" with one or
more tags as known in the art. Such tags may then be targets for
higher order ligands. Exemplary tags include biotin, digoxygenin,
His-Tag, Glutathion-S-Transferase, FLAG, GFP, myc-tag, influenza A
virus haemagglutinin (HA), maltose binding protein, and the like.
In the case of a peptide or polypeptide, the tag may be at (or in
proximity to) the N-terminus and/or C-terminus. Exemplary labels
include labels detectable by an appropriate detection method.
Exemplary labels include gold particles, latex beads, acridan
ester, luminol, ruthenium, enzymatically active labels, radioactive
labels, magnetic labels ("e.g. magnetic beads", including
paramagnetic and superparamagnetic labels), and fluorescent labels.
Enzymatically active labels are also within the scope of the
instant disclosure, and exemplary enzymatically active labels
include horseradish peroxidase, alkaline phosphatase,
beta-Galactosidase, Luciferase, and derivatives thereof. Suitable
substrates for detection include di-amino-benzidine (DAB),
3,3'-5,5'-tetramethylbenzidine, NBT-BCIP (4-nitro blue tetrazolium
chloride and 5-bromo-4-chloro-3-indolyl-phosphate, for example as
ready-made stock solution from Roche Diagnostics), CDP-Star.TM.
(for example as available from Amersham Biosciences), ECF.TM. (for
example as available from Amersham Biosciences). A suitable
enzyme-substrate combination may result in a colored reaction
product, fluorescence or chemoluminescence, which can be measured
according to various methods known in the art such as using a
light-sensitive film or a suitable camera system). As for measuring
the enzymatic reaction, the various methods given above apply
analogously. Exemplary fluorescent labels include fluorescent
proteins (such as GFP and its derivatives), Cy3, Cy5, Texas Red,
Fluorescein, and the Alexa dyes (e.g. Alexa 568). Additionally,
further fluorescent labels are available, for example, from
Molecular Probes (for example as available from Oregon). Also the
use of quantum dots as fluorescent labels is contemplated.
Exemplary radioactive labels include .sup.35S, .sup.125I, .sup.32P,
.sup.33P and the like. A radioactive label may be detected by any
method known including, for example, a light-sensitive film or a
phosphor imager. Exemplary measurement methods according the
present invention also include precipitation (e.g.,
immunoprecipitation), electrochemiluminescence (e.g.,
electro-generated chemiluminescence), RIA (e.g., radioimmunoassay),
ELISA (e.g., enzyme-linked immunosorbent assay), sandwich enzyme
immune tests, electrochemiluminescence sandwich immunoassays (e.g.,
ECLIA), dissociation-enhanced lanthanide fluoro immuno assay (e.g.,
DELFIA), scintillation proximity assay (e.g., SPA), turbidimetry,
nephelometry, latex-enhanced turbidimetry or nephelometry, or solid
phase immune tests. Further methods known in the art (such as gel
electrophoresis, 2D gel electrophoresis, SDS polyacrylamid gel
electrophoresis (SDS-PAGE), Western Blotting, and mass
spectrometry), may also be used alone or in combination with
labelling or other detection methods as described above.
[0050] According to some embodiments, the amount of a peptide or
polypeptide may be determined as follows: (a) contacting a solid
support comprising a ligand for the peptide or polypeptide as
specified above with a sample comprising the peptide or polypeptide
and (b) measuring the amount of the peptide or polypeptide which is
bound to the support. In some exemplified embodiments, the ligand
may be chosen from the group consisting of nucleic acids, peptides,
polypeptides, antibodies and aptamers, may be present on a solid
support in immobilized form. Materials for manufacturing solid
supports are known in the art and include, for example,
commercially available column materials, polystyrene beads, latex
beads, magnetic beads, colloid metal particles, glass and/or
silicon chips and surfaces, nitrocellulose strips, membranes,
sheets, duracytes, wells and walls of reaction trays, plastic tubes
etc. The ligand or agent may be bound to many different carriers.
Examples of well-known carriers include glass, polystyrene,
polyvinyl chloride, polypropylene, polyethylene, polycarbonate,
dextran, nylon, amyloses, natural and modified celluloses,
polyacrylamides, agaroses, and magnetite. The nature of the carrier
may be either soluble or insoluble, according to the disclosure.
Methods for fixing/immobilizing said ligand are also known and
include, but are not limited to ionic, hydrophobic, covalent
interactions and the like. It is also contemplated to use
"suspension arrays" as arrays according to the present disclosure
(see, for example, Nolan 2002, Trends Biotechnol. 20(1):9-12). In
various embodiments of suspension arrays, the carrier (for example,
a microbead or microsphere) may be present in suspension. The array
may consist of different microbeads or microspheres, possibly
labeled, carrying different ligands. Methods of producing such
arrays, for example based on solid-phase chemistry and photo-labile
protective groups, are known (see for example U.S. Pat. No.
5,744,305).
[0051] The term "amount" as used herein encompasses an absolute
amount of a polypeptide or peptide, the relative amount (or
concentration) of the polypeptide or peptide, and any value or
parameter which correlates thereto or can be derived therefrom.
Such values or parameters comprise intensity signal values from all
specific physical or chemical properties obtained from the said
peptides by direct measurements, for example intensity values in
mass spectra or NMR spectra. Moreover, encompassed are all values
or parameters which are obtained by indirect measurements specified
elsewhere in this description, such as response levels determined
from biological read out systems in response to the peptides or
intensity signals obtained from specifically bound ligands. Values
correlating to the aforementioned amounts or parameters may also be
obtained by all standard mathematical operations.
[0052] As used herein, the term "sample" may refer to a sample of a
body fluid, a sample of separated cells, or even a sample from a
tissue or an organ. Samples of body fluids may be obtained
according to any known techniques and include, for example, samples
of blood, plasma, serum, urine, or buccal swabs. Tissue or organ
samples may be obtained from any tissue or organ, for example, such
as by biopsy. Separated cells may be obtained from the body fluids
or the tissues or organs by separating techniques such as
centrifugation or cell sorting. According to embodiments of the
instant disclosure, cell-, tissue- or organ samples are obtained
from those cells, tissues, or organs which express or produce the
peptides referred to herein.
[0053] The term "patient" as used in the present application may
refer to a mammal, such as a human. The patient may suffer from a
medical event and may present with the above described condition at
an emergency department, for example. According to some
embodiments, the patient may be an emergency patient, i.e., a
patient whose condition requires immediate medical attention and/or
intensive care. In some cases, such a patient may have been
apparently healthy with respect to acute circulatory and acute
ischemic events prior to the onset of an acute medical event. Thus,
in some embodiments, the patient's vital functions may not have
been monitored closely or even at all before arrival at the
emergency department. Thus, in some embodiments, little
retrospective information may be available about the cause of the
life threatening condition of the patient when he presents at the
emergency department. In such situations and embodiments, the
method of the present disclosure, rapidly performed at the point of
care, is helpful as a first indicator of the cause of the patient's
condition and for guiding further (in some cases more specific)
diagnostic measures.
[0054] Exemplary "further" diagnostic measures may include standard
laboratory diagnostic procedures such as measuring creatinin,
glucose, electrolyte, liver enzymes; total blood cell count; blood
gas analysis; and imaging (such as ECG, echocardiography, computer
tomography, and angiography) for example.
[0055] The term "comparing" as used herein encompasses comparing
the amount of the peptide or polypeptide comprised by the sample to
be analyzed with an amount of a suitable reference source specified
elsewhere in this description. It is to be understood that
comparing as used herein refers to a comparison of corresponding
parameters or values, such as an absolute amount compared to an
absolute reference amount while a concentration is compared to a
reference concentration or an intensity signal obtained from a test
sample is compared to the same type of intensity signal of a
reference sample, for example. The comparison referred to in step
(c) of the method of the present invention may be carried out
manually or computer assisted, for example. For a computer assisted
comparison, the value of the determined amount may be compared to
values corresponding to suitable references which are stored in a
database by a computer program. The computer program may further
evaluate the result of the comparison, i.e. automatically provide
the desired assessment in a suitable output format. Based on the
comparison of the amounts determined in steps a) and b) and the
reference amounts of the method of the present invention, it is
possible to predict the risk of the subject of suffering of one or
more of the complications referred to herein. Therefore, the
reference amount may be chosen so that either a difference or a
similarity in the compared amounts allows identifying those
patients whose condition is caused by an acute circulatory event,
by an ischemic event, by both kinds of events or by none of these
events.
[0056] The term "reference amount" as used herein refers to an
amount which allows diagnosing whether a patient suffered from an
acute circulatory event, an ischemic event, both kinds of event or
none of them. Accordingly, the reference may be derived from any of
(i) a patient known to have suffered from the respective kind of
event or (ii) a patient known to have not suffered from the
respective event, for example. Moreover, the reference amount may
define a threshold amount, whereby an amount larger than the
threshold is indicative for a subject which has suffered one or
both of the above mentioned events. The reference amount applicable
for an individual subject may vary depending on various
physiological parameters such as age, gender, or subpopulation, as
well as on the means used for the determination of the polypeptide
or peptide referred to herein. A suitable reference amount may be
determined by the method of the present invention from a reference
sample to be analyzed together, i.e. simultaneously or
subsequently, with the test sample. A preferred reference amount
serving as a threshold may be derived from the upper limit of
normal (ULN), for example the upper limit of the physiological
amount found in a population of subjects who have not suffered or
are not suffering from the complications as defined above. The ULN
for a given population of subjects may be determined by various
known techniques. A suitable technique, for example, may be to
determine the median of the population for the peptide or
polypeptide amounts to be determined in the method of the present
invention.
[0057] Reference amounts of a diagnostic marker (e.g., of ANP-type
peptide or sFlt-1) can be established, and the level of the marker
in a patient sample may be compared to the reference amount. The
sensitivity and specificity of a diagnostic and/or prognostic test
depends on more than just the analytical "quality" of the test, for
example, they also depend on how abnormal results are identified
(or characterized). The distribution of the measured amounts of the
markers of the present invention, in a population of patients
suffering or having suffered from a circulatory and/or ischemic
complication, may be compared to the distribution of the amounts of
the markers in patients without the complication(s). Statistical
methods known to a person of skill in the art can be used to define
a threshold amount that may be used to separate patients having
suffered from one or both of the said complications and patients
not having suffered from said complications. For example, the
calculation of Receiver Operating Characteristic curves, or "ROC"
curves may be used for this purpose. ROC-curves are typically
calculated by plotting the value of a variable versus its relative
frequency in "normal" and "disease" populations. For example, with
any particular marker, a distribution of marker levels for subjects
with and without a disease may (and even likely will) overlap.
Under such conditions, a test does not absolutely distinguish
normal from disease with 100% accuracy, and the area of overlap may
indicate where the test cannot distinguish normal from disease. A
threshold may be selected, above which (or below which, depending
on how a marker changes with the disease) the test is considered to
be abnormal and below which the test is considered to be normal.
The area under the ROC curve is a measure of the probability that
the perceived measurement will allow correct identification of a
condition. ROC curves can be used even when test results do not
necessarily give an accurate number. As long as one can rank
results, one can create an ROC curve. For example, results of a
test on "disease" samples might be ranked according to degree (say
l=low, 2=normal, and 3=high). This ranking can be correlated to
results in the "normal" population, and a ROC curve created. These
methods are known in the art, for example, see Hanley et al,
Radiology 143: 29-36 (1982).
[0058] In certain embodiments, markers (for example, ANP-type
peptide and sFlt-1) are selected to exhibit at least about 70%
sensitivity and in some case at least about 80% sensitivity, at
least about 85% sensitivity, at least about 90% sensitivity, and
even at least about 95% sensitivity, combined with at least about
70% specificity, at least about 80% specificity, at least about 85%
specificity, at least about 90% specificity, or even at least about
95% specificity. In exemplary embodiments, both the sensitivity and
specificity are at least about 75%, at least about 80%, at least
about 85%, at least about 90%, and even at least about 95%. The
term "about" in this context refers to +/-5% of a given
measurement.
[0059] The reference amount for NT-proANP comprises about 1000
pg/ml, about 2000 pg/ml, about 2500 pg/ml, about 3000 pg/ml, about
4000 pg/ml or about 5000 pg/ml. According to some exemplified
embodiments, reference amount for NT-proANP comprises about 2500
pg/ml. According to such embodiments, amounts larger than this
amount are indicative of a patient having suffered from a
circulatory complication.
[0060] If the circulatory complication is an arrhythmia, the
reference amount for NT-proANP may comprise about 1000 pg/ml, about
2000 pg/ml, about 2500 pg/ml, about 3000 pg/ml, about 4000 pg/ml or
about 5000 pg/ml, for example. According to some exemplified
embodiments the reference amount for NT-proANP comprises about 2500
pg/ml. If the circulatory complication is tachycardia, the
reference amount may comprise about 1000 pg/ml, about 2000 pg/ml,
about 2500 pg/ml, about 3000 pg/ml, about 4000 pg/ml or about 5000
pg/ml. According to some exemplified embodiments, the reference
amount comprises about 2500 pg/ml.
[0061] The reference amount for sFlt-1 comprises about 100 pg/ml,
about 200 pg/ml, about 300 pg/ml, about 400 pg/ml, about 500 pg/ml,
about 750 pg/ml or about 1000 pg/ml. According to some exemplified
embodiments, the reference amount comprises about 500 pg/ml.
According to such embodiments, amounts larger than this amount are
indicative of a patient having suffered an ischemic
complication.
[0062] The term "about" is meant to indicate +/-30% of the
indicated amount. In some embodiments of the instant disclosure,
the term "about" is indicative of +/-20% of the indicated amount or
even +/-10% of the indicated amount or even +/-5% of the indicated
amount.
[0063] Increased levels of sFlt-1 and the ANP-type peptide are
indicative of a combined circulatory and ischemic complication. The
reference amounts cited above for sFlt-1 and the ANP-type peptide
also apply for combined circulatory and ischemic complications,
including those for particular circulatory complications like
arrhythmia and tachycardia.
[0064] In view of the above explanations and examples, a reference
value for an ANP-type peptide other than NT-proANP, may be
determined allowing for diagnosis of a pathophysiological cause of
an acute medical event in an emergency patient is a circulatory
complication.
[0065] Embodiments of the method of the present invention allows
for a rapid diagnosis and guidance for further
examination/diagnosis aiming at establishing the exact cause or
causes underlying the pathophysiological state of patients
suffering from an acute medical event or aiming at establishing the
disease or condition which the patient suffers from. For example,
by indicating a cause underlying an organ failure the method is
able to guide the further diagnosis. Thus, for example, it is
possible to exclude certain diseases by differential diagnosis. The
reduced time for diagnosis enables a more rapid onset of causal
treatment and, thus, reduces the suffering of the patient, may
prevent a further deterioration of the patient's condition or even
death, for example. The method of the present invention also may be
performed at the bed side, thus leading the physician early and
quickly to the other required diagnostic measures.
[0066] For example, embodiments of methods of the present invention
may also indicate not only the cause, but also the extent or
severity of a circulatory or ischemic complication. Complications
of greater extent or severity may be associated with higher amounts
of the ANP-type peptide and/or sFlt-1 as compared to complications
of lesser extent or severity. For example, a reference amount may
be determined in a patient with a complication of known extent or
severity. Comparison of a sample from a patient suffering from or
having suffered from a complication of unknown extent or severity
to a reference amount derived from a patient or collective of
patients having suffered a complication of known severity makes it
possible to establish the extent or severity of said patient's
complication quickly. For example, amounts of the ANP-type peptide
and/or sFlt-1 that are higher than the reference amount may
indicate a more severe or extended complication. Criteria for the
extent and/or severity of a condition, include its duration (e.g.,
a longer period of cardiac arrhythmia), or the intensity of the
complication (e.g., the amount of ischemic tissue).
[0067] Furthermore, embodiments of the present invention may relate
to a device for, or adapted for, assessing (including rapidly
assessing) if the pathophysiological cause of an acute medical
event in an emergency patient is a circulatory and/or an ischemic
complication. Exemplary embodiments may comprise: [0068] a) means
for determining the amount of an ANP-type peptide in a sample of a
patient; [0069] b) means for determining the amount of sFlt-1 in a
sample from a patient; [0070] c) means for comparing the measured
amounts of the ANP-type peptide and sFlt-1 to reference amounts;
and [0071] d) means for establishing a diagnosis based on the
results of the comparison.
[0072] The term "device" as used herein relates to a system of
means comprising at least the aforementioned means operatively
linked to each other as to allow the prediction. Exemplary means
for determining the amounts of an ANP-type peptide and sFlt-1, and
means for carrying out the comparison are disclosed above in
connection with the method of the invention. Exemplary methods for
linking the means in an operating manner will depend on the type of
means included in the device. For example, where means for
automatically determining the amount of the peptides are applied,
the data obtained by said automatically operating means can be
processed by a computer program, for example, in order to obtain
the desired results. The means may comprise a single device in such
a case, for example. Said device may accordingly include an
analyzing unit for the measurement of the amount of the peptides or
polypeptides in an applied sample and a computer unit for
processing the resulting data for the evaluation. Alternatively,
where means such as test strips are used for determining the amount
of the peptides or polypeptides, the means for comparison may
comprise control strips or tables allocating the determined amount
to a reference amount. The test strips may be coupled to a ligand
which specifically binds to the peptides or polypeptides referred
to herein, for example. The strip or device may comprise means for
detection of the binding of said peptides or polypeptides to the
said ligand. Exemplary means for detection are disclosed in
connection with embodiments relating to the method of the invention
above. In such a case, the means are operatively linked in that the
user of the system brings together the result of the determination
of the amount and the diagnostic or prognostic value thereof due to
the instructions and interpretations given in a manual. The means
may appear as separate devices in such an embodiment and may be
packaged together as a kit. A person of skill in the art will
understand how to link the means. Preferred devices are those which
can be applied without the particular knowledge of a specialized
clinician (for example, test strips or electronic devices which
merely require loading with a sample). The results may be given as
output of raw data which need interpretation by the clinician as
processed information (i.e. evaluated raw data, the interpretation
of which does not require a clinician). Further exemplary devices
comprise the analyzing units/devices (such as, biosensors, arrays,
solid supports coupled to ligands specifically recognizing the
peptide, Plasmon surface resonance devices, NMR spectrometers,
mass-spectrometers, etc.) and/or evaluation units/devices referred
to above in accordance with methods described and disclosed
herein.
[0073] Additionally, as disclosed above, according to some
embodiments of the present disclosure, a kit for rapidly assessing
if the pathophysiological cause of an acute medical event in an
emergency patient is a circulatory and/or an ischemic complication
is provided. Such embodiments may comprise: [0074] a) means for
determining the amount of a ANP-type peptide in a sample of a
patient; [0075] b) means for determining the amount of sFlt-1 in a
sample from a patient; [0076] c) means for comparing the measured
amounts of the ANP-type peptide and sFlt-1 to reference amounts;
and [0077] d) means for establishing a diagnosis based on the
results of the comparison.
[0078] As used herein, the term "kit" may refer to a collection of
the aforementioned means. For example, such means may be provided
within a separate container or within a single container. The
container may also comprise instructions for carrying out the
methods disclosed and described herein.
[0079] According to other embodiments of the instant disclosure,
methods for diagnosis of a vascular disease with atherosclerotic
etiology are provided. Such embodiments may comprise the steps of:
[0080] a) determining the amount of an ANP-type peptide in a sample
of a patient; [0081] b) determining the amount of sFlt-1 in a
sample from a patient; [0082] c) comparing the amounts measured in
steps a) and b) to reference amounts; and [0083] d) establishing a
diagnosis based on the results of c).
[0084] According to some embodiments, the instant disclosure
relates to a method for diagnosing whether a diabetes patient is
suffering from a cardiovascular complication or is at risk of
suffering from a cardiovascular complication. Such embodiments may
comprise the steps of: [0085] a) measuring, preferably in vitro,
the level(s) of at least one cardiac hormone (e.g. NT-proBNP) in a
sample from the patient; and [0086] b) diagnosing the
cardiovascular complication or the risk of suffering from
cardiovascular complication by comparing the measured level(s) to
known level(s) associated with the cardiovascular complication or
the risk.
[0087] Moreover, a method for diagnosing whether a diabetes patient
is suffering from a microangiopathy or is at risk of suffering from
a microangiopathy is disclosed herein. According to some
embodiments, this method comprises the steps of: [0088] a)
measuring in vitro the level(s) of PLGF or PLGF-1 variant in blood
serum, blood or blood plasma sample from the patient; and [0089] b)
diagnosing microangiopathy or the risk of suffering from
microangiopathy by comparing the measured level(s) to known
level(s) associated with microangiopathy or the risk.
[0090] The following examples, sequence listing, and figures are
provided for the purpose of demonstrating various embodiments of
the instant disclosure and aiding in an understanding of the
present disclosure, the true scope of which is set forth in the
appended claims. These examples are not intended to, and should not
be understood as, limiting the scope or spirit of the instant
disclosure in any way. It should also be understood that
modifications can be made in the procedures set forth without
departing from the spirit of the disclosure.
EXAMPLES
Example 1
[0091] In 403 patients (median age: 52.4 years) with life
threatening conditions which required intensive care sFlt-1 and
NT-proANP were determined within 4 hours after hospitalization. At
the time of sampling all patients were mechanically ventilated.
Their circulation was stable.
[0092] NT-proANP was determined with the proANP ELISA assay
(BI-20892) obtained from Biomedica, Vienna, Austria. This sandwich
assay comprises a polyclonal sheep NT-proANP specific antibody
bound to a microtiterstrip. The sample is added to the
microtiterstrip so that the proANP can bind to the antibody. After
binding of the proANP to the first antibody a second proANP
specific antibody is added to the vessel. This second antibody is
conjugated with horseradish peroxidase (HRP). After incubation the
unbound enzyme-conjugated antibody is removed by washing the
microtiterstrip. Finally, tetramethylbenzidine (TMB) is added as a
substrate for the HRP. The more proANP the sample contains, the
more conjugated antibody binds. Thus, the total activity of HRP
present in the vessel depends on the amount of proANP in the sample
and the initial rate of TMB converted is a measure for the amount
of NT-proANP in the sample.
[0093] sFlt-1 was determined with a sFlt-1 immunoassay to be used
with the Elecsys and COBAS analyzers from Roche Diagnostics,
Mannheim, Germany. The assay is based on the sandwich principle and
comprises two monoclonal sFlt-1 specific antibodies. The first of
these is biotinylated and the second one is labelled with a
Tris(2,2'-bipyridyl)ruthenium(II)-complex. In a first incubation
step both antibodies are incubated with the sample. A sandwich
complex comprising sFlt-1 and the two different antibodies is
formed. In a next incubation step streptavidin-coated beads are
added to this complex. The beads bind to the sandwich complexes.
The reaction mixture is then aspirated into a measuring cell where
the beads are magnetically captured on the surface of an electrode.
The application of a voltage then induces a chemiluminescent
emission from the ruthenium complex which is measured by a
photomultiplier. The amount of light is dependent on the amount of
sandwich complexes on the electrode.
[0094] Tachycardia was determined by electrocardiography or by
measuring the pulse of the patient for at least 30 seconds.
TABLE-US-00001 TABLE 1 Circulatory events. Patients NT-proANP
Patients with with [pg/ml] N* arrhythmia.sup..sctn. [%]
tachycardia** [%] <1000 64 0 0.0 12 18.8 1000 to 2500 204 16 0.8
101 49.5 2500 to 5000 97 32 33.0 59 60.8 5000 to 10,000 35 21 60.0
28 80.0 >10,000 3 1 33.3 3 100.0 *Total number of patients
**Tachycardia was diagnosed if the patient exhibited a pulse rate
of >120 bpm. .sup..sctn.Arrhythmia was defined as absolute
arrhythmia or more than 10 extra heart beats per minute
TABLE-US-00002 TABLE 2 Proportion of patients with ischemic events
(systolic BP <80 mmHg). Ischemic sFlt1 [pg/ml] N* events** [%]
<100 229 2 0.0 100 to 500 140 11 0.8 500 to 1000 4 1 25.0 1000
to 5000 9 6 66.7 >5000 21 13 61.9 *Total number of patients
**Number of patients.
[0095] Patients were considered to suffer from an ischemic event if
the following criteria were met: organ specific pain, reduced pulse
in the affected area and paleness of the skin.
[0096] Table 1 shows that with increasing amounts of NT-proANP the
proportion of patients with circulatory events increased. More than
33% of patients with NT-proANP levels above about 2500 pg/ml had
suffered before sampling or still suffered at the time of sampling
from arrhythmia and more than 60% of the patients with NT-proANP
levels above 2500 pg/ml had suffered before sampling or still
suffered at the time of sampling from tachycardia. Thus, by
measuring the NT-proANP level in the emergency patients,
circulatory complications, as exemplified by arrhythmia and
tachycardia may be reliably diagnosed. To the surprise of the
inventors, such diagnosis may be accomplished even within 4 h after
hospitalization.
[0097] Table 2 shows the prevalence of ischemic events. More than
25% of the patients with sFlt-1 levels above about 500 pg/ml had
suffered before sampling, or still suffered at the time of
sampling, from a systolic blood pressure below 80 mmHg.
[0098] In view of the above, measuring the sFlt-1 level in the
emergency patients allows for safely and rapidly diagnosing
ischemic complications.
[0099] Each of the two markers sFlt-1 and NT-proANP (or another
ANP-type peptide) provides for a statistically independent
diagnostic measure. By combining the measurement of sFlt-1 and
NT-proANP (or another ANP-type peptide) into one diagnostic test, a
physician may readily and quickly determine the pathophysiological
cause of an acute medical event in an emergency patient, such as if
the given emergency patient suffers from a circulatory and/or an
ischemic complication.
Illustrative Embodiments
[0100] 1. A method for rapidly diagnosing if an acute medical event
in an emergency patient is associated with a circulatory and/or an
ischemic complication, comprising the steps of [0101] a)
determining the amount of an ANP-type peptide in a sample of a
patient; [0102] b) determining the amount of sFlt-1 in a sample
from a patient; [0103] c) comparing the amounts measured in steps
a) and b) to reference amounts; and establishing a diagnosis based
on the results of c), [0104] d) establishing a diagnosis based on
the results of c), wherein an increased level of the ANP-type
peptide relative to the reference amount is indicative of a
circulatory complication and wherein an increased level of sFlt-1
relative to the reference amount is indicative of an ischemic
complication, [0105] wherein the circulatory complication is caused
by cardiac arrhythmia, and [0106] wherein the ischemic complication
is characterized by a systolic blood pressure of less than 80 mmHg.
[0107] 2. The method of 1, wherein increased levels of sFlt-1 and
the ANP-type peptide relative to the reference amounts are
indicative of a combined circulatory and ischemic complication.
[0108] 3. The method of 1, wherein the acute medical event is heart
failure, lung failure or renal failure. [0109] 4. The method of 2,
wherein the ischemic complication occurs in the spleen, in the
heart, in the kidney, in the bowel or in the limbs. [0110] 5. The
method of 1, wherein the ANP-type peptide is NT-proANP. [0111] 6.
The method of 5, wherein the reference amount for NT-proANP is
about 2500 pg/ml. [0112] 7. The method of 1, wherein the reference
amount for sFlt-1 is about 500 pg/ml. [0113] 8. A device for
rapidly diagnosing if an acute medical event in an emergency
patient is associated with a circulatory and/or an ischemic
complication, comprising [0114] a) analyzing unit for determining
the amount of an ANP-type peptide in a sample of a patient; [0115]
b) analyzing unit for determining the amount of sFlt-1 in a sample
from a patient; [0116] c) computer unit for comparing the measured
amounts of the ANP-type peptide and sFlt-1 to reference amounts;
and [0117] d) computer unit for establishing a diagnosis based on
the results of the comparison, wherein an increased level of the
ANP-type peptide relative to the reference amount is indicative of
a circulatory complication and wherein an increased level of sFlt-1
relative to the reference amount is indicative of an ischemic
complication, and [0118] wherein the circulatory complication is
caused by cardiac arrhythmia, and [0119] wherein the ischemic
complication is characterized by a systolic blood pressure of less
than 80 mmHg. [0120] 9. The device of 8, wherein the ANP-type
peptide is proANP. [0121] 10. A kit for rapidly diagnosing if an
acute medical event in an emergency patient is associated with a
circulatory and/or an ischemic complication, said kit comprising
instructions for carrying out the diagnosis, and further comprising
[0122] a) analyzing unit for determining the amount of an ANP-type
peptide in a sample of a patient; [0123] b) analyzing unit for
determining the amount of sFlt-1 in a sample from a patient; [0124]
c) computer unit for comparing the measured amounts of the ANP-type
peptide and sFlt-1 to reference amounts; and [0125] d) computer
unit for establishing a diagnosis based on the results of the
comparison, wherein an increased level of the ANP-type peptide
relative to the reference amount is indicative of a circulatory
complication and wherein an increased level of sFlt-1 relative to
the reference amount is indicative of an ischemic complication
[0126] wherein the circulatory complication is caused by cardiac
arrhythmia, and [0127] wherein the ischemic complication is
characterized by a systolic blood pressure of less than 80 mmHg.
[0128] 11. The kit of 9, wherein the ANP-type peptide p-reference
is proANP.
[0129] All publications, patents and applications are hereby
incorporated by reference in their entirety to the same extent as
if each such reference was specifically and individually indicated
to be incorporated by reference in its entirety.
[0130] While this disclosure has been described as having an
exemplary design, the present disclosure may be further modified
within the spirit and scope of this disclosure. This application is
therefore intended to cover any variations, uses, or adaptations of
the disclosure using its general principles. Further, this
application is intended to cover such departures from the present
disclosure as come within the known or customary practice in the
art to which this disclosure pertains.
* * * * *