U.S. patent application number 15/216980 was filed with the patent office on 2016-11-10 for prediction of postpartum hellp syndrome, postpartum eclampsia or postpartum preeclampsia.
The applicant listed for this patent is Roche Diagnostics Operations, Inc.. Invention is credited to Thomas Dieterle, Martin Hund, Olav Lapaire.
Application Number | 20160327564 15/216980 |
Document ID | / |
Family ID | 49998135 |
Filed Date | 2016-11-10 |
United States Patent
Application |
20160327564 |
Kind Code |
A1 |
Hund; Martin ; et
al. |
November 10, 2016 |
PREDICTION OF POSTPARTUM HELLP SYNDROME, POSTPARTUM ECLAMPSIA OR
POSTPARTUM PREECLAMPSIA
Abstract
The present invention is directed to a method for predicting the
risk of a female subject to develop postpartum HELLP syndrome,
postpartum preeclampsia, or postpartum eclampsia. The method is
based on the determination of the levels of i) sFlt-1 and PlGF, or
ii) Endoglin and PlGF in a first sample obtained from said subject
before delivery of baby, and a second sample of from said subject
obtained after delivery of baby. Moreover, encompassed by the
invention are devices and kits for carrying out the method of the
present invention.
Inventors: |
Hund; Martin; (Horw, CH)
; Dieterle; Thomas; (Freiburg, DE) ; Lapaire;
Olav; (Binningen, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Roche Diagnostics Operations, Inc. |
Indianapolis |
IN |
US |
|
|
Family ID: |
49998135 |
Appl. No.: |
15/216980 |
Filed: |
July 22, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/EP2015/051457 |
Jan 26, 2015 |
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15216980 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2800/368 20130101;
G01N 2333/515 20130101; G01N 2800/50 20130101; G01N 33/689
20130101; G01N 2333/91205 20130101; G01N 33/6863 20130101; G01N
2333/705 20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 24, 2014 |
EP |
14152447.0 |
Claims
1. A method for predicting the risk of a female subject to develop
at least one preeclampsia related adverse outcome after delivery of
baby, said method comprising the steps of a) measuring in a first
sample obtained from a female subject with an uneventful pregnancy
before delivery of baby i) the level of the biomarker sFlt-1
(soluble fms-like tyrosine kinase-1) or the level of the biomarker
Endoglin, and ii) the level of the biomarker PlGF (Placental Growth
Factor), b) calculating a first ratio of the levels of the
biomarkers as measured in step a), c) measuring in a second sample
obtained from said female subject after delivery of baby the levels
of the biomarkers as measured in step a), d) calculating a second
ratio of the levels measured in step c), and e) comparing the
second ratio to the first ratio.
2. The method of claim 1, wherein in steps a) and c) the levels of
the biomarkers sFlt-1 and PlGF are measured.
3. The method of claim 2, wherein the at least one preeclampsia
related adverse outcome is selected from the group consisting of
postpartum preeclampsia, postpartum eclampsia and postpartum HELLP
syndrome.
4. The method of claim 1, wherein the female subject with an
uneventful pregnancy did not exhibit preeclampsia, severe
preeclampsia, eclampsia and/or a HELLP syndrome before delivery of
baby.
5. The method of claim 1, wherein the first sample has been
obtained within 48 hours before delivery of baby.
6. The method of claim 1, wherein the second sample has been
obtained within 24 hours after delivery of baby.
7. The method of claim 1, wherein the first and the second ratio
are the ratios of sFlt-1 to PlGF, or of Endoglin to PlGF, and
wherein an increase of the second ratio, or an essentially
unchanged second ratio as compared to the first ratio is indicative
for a subject who is at risk of developing at least one
preeclampsia related adverse outcome after delivery of baby, and/or
wherein a decrease of the second ratio as compared to the first
ratio is indicative for a subject who is not at risk of developing
a preeclampsia related adverse outcome after delivery of baby.
8. The method of claim 1, wherein the first and the second ratio
are the ratios of PlGF to sFlt-1, or of PlGF to Endoglin, and
wherein decrease of the second ratio or an essentially unchanged
second ratio as compared to the first ratio is indicative for a
subject who is at risk of developing at least one preeclampsia
related adverse outcome after delivery of baby, and/or wherein an
increase of the second ratio as compared to the first ratio is
indicative for a subject who is not at risk of developing a
preeclampsia related adverse outcome after delivery of baby.
9. The method of claim 1, wherein the subject is human.
10. The method of claim 1, wherein the sample is a blood, serum or
plasma sample, or wherein the sample is a urine sample.
11. The method of claim 1, wherein the risk to develop at least one
preeclampsia related adverse outcome within seven days after
delivery of baby is predicted.
12. A device adapted for predicting the risk of a female subject to
develop at least one preeclampsia related adverse outcome after
delivery of baby, said device comprising: a) an analyzer unit
comprising an agent which specifically binds to sFlt-1 and/or
Endoglin, and an agent which specifically binds to PlGF, said unit
being adapted for measuring the level of sFlt-1 and/or Endoglin and
the level of PlGF in a first sample of a female subject obtained
before delivery of baby and a second sample of said female subject
obtained after delivery of baby; and b) an evaluation unit
comprising a data processor having implemented an algorithm for
carrying out the following steps of: i) calculating a first ratio
from said levels of sFlt-1 or Endoglin and PlGF determined in the
first sample and a second ratio from said levels of sFlt-1 or
Endoglin and PlGF determined in the second sample; and ii)
comparing the value of the said first and the said second ratio,
and iii) predicting the risk of said subject to develop at least
one preeclampsia related adverse outcome after delivery of baby.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of International Patent
Application No. PCT/EP2015/051457 filed Jan. 26, 2015, and claims
priority to EP 14152447.0 filed Jan. 24, 2014, the disclosures of
which are hereby incorporated by reference in their entirety.
BACKGROUND
[0002] The present invention is directed to a method for predicting
the risk of a female subject with an uneventful pregnancy to
develop at least one preeclampsia related adverse outcome after
delivery of baby. The method is based on the determination of the
levels of i) sFlt-1 and PlGF, or ii) Endoglin and PlGF in a first
sample obtained from said subject before delivery of baby, and a
second sample of from said subject obtained after delivery of baby.
Moreover, encompassed by the invention are devices and kits for
carrying out the method of the present invention.
[0003] Pregnancy may be complicated in different ways. It is on one
hand associated with pregnancy related mortality of the pregnant
woman and, on the other hand, also associated with increased
morbidity and mortality of the newborn. Maternal mortality at a
rate of 14.5 per 100,000 live births, is more frequent in pregnant
women above the age of 39 years and may be caused by hemorrhage,
thrombotic pulmonary embolism, infections, cardiomyopathy and
cardiovascular and noncardiovascular conditions as well as
hypertensive disorders among which preeclampsia is the most
frequent (Berg 2010, Obstetrics and Gynecology: 116:
1302-1309).
[0004] Preeclampsia complicates approximately 2 to 8 percent of all
pregnancies and is a major contributor to maternal and fetal
mortality worldwide (Duley 2009, Semin Perinatol: 33: 130-37).
Preeclampsia usually occurs during pregnancy. However, it may also
develop postpartum, i.e. after delivery of baby.
[0005] Preeclampsia is generally defined as pregnancy associated or
induced hypertension. It is characterized by hypertension and
proteinuria. Details are also found in the standard text books of
medicine and the Guidelines of the various clinical societies,
e.g., Brown M A, Lindheimer M D, de Swiet M, Van Assche A, Moutquin
J M: The classification and diagnosis of the hypertensive disorders
of pregnancy: statement from the International Society for the
Study of Hypertension in Pregnancy (ISSHP). Hypertens Pregnancy
2001, 20:IX-XIV or ACOG Practice Bulletin, Clinical Management
Guidelines for Obstetrician--Gynecologists, no.: 33, January 2002
or DGGG. S1-Leitlinie: Diagnostik und Therapie hypertensiver
Schwangerschaftserkrankungen der Deutschen Gesellschaft fur
Gynakologie und Geburtshilfe, AWMF online, AWMF Register Nummer
015/018, Klasse S1.
[0006] In addition to preeclampsia, there are further preeclampsia
related adverse outcomes which may develop after childbirth, e.g.
HELLP syndrome and eclampsia. All conditions are associated with
adverse outcomes for the mother postpartum.
[0007] HELLP syndrome is a life-threatening obstetric complication
and involves hemolytic anemia, elevated liver function tests
(LFTs), and low platelet count. HELLP usually begins during the
third trimester; however up to 30% of all patients develop this
syndrome after parturition, typically within 48 hours.
Unexpectedness, suddenness, and fulminant course of this syndrome
are essential. In 20% of cases there maybe no evidence of
pre-eclampsia before or during labour and all laboratory findings
were normal. (Haram K, Svendsen E, Abildgaard U. The HELLP
syndrome: clinical issues and management. A review. BMC Pregnancy
and Childbirth 2009; 9(8). http://dx.doi.org/10.1186/1471-2393-9-8;
Pop-Trajkovic et al. 2013 Uppsala Journal of Medical Sciences 118,
51-53).
[0008] Eclampsia is commonly defined as new onset of grand mal
seizure activity and/or unexplained coma during pregnancy or
postpartum in a woman with signs or symptoms of preeclampsia. It
typically occurs during or after the 20th week of gestation or in
the postpartum period after childbirth and delivery of the
placenta.
[0009] There is a high unmet medical need to identify women at risk
of developing postpartum HELLP syndrome, eclampsia, or preeclampsia
immediately after birth.
[0010] Placental growth factor (PlGF), soluble Endoglin und soluble
fms-like tyrosine kinase 1 (sFlt-1) have been described as marker
for diagnosing and prediction preeclampsia during pregnancy (see
e.g. WO2004/008946, WO2008/034750; Rana, 2007, Hypertension
50:137-142). Ratios of sFlt-1 and PlGF or Endoglin and PlGF have
been reported as diagnostic or prognostic parameters for
preeclampsia in pregnant women before delivery.
[0011] It is known in the literature that angiogenic factors and
anti-angiogenic factors rapidly decline after delivery in healthy
women as well as in preeclamptic women.
[0012] Wikstrom et al. examined the concentration levels of sFlT-1
and PlGF before and after delivery in preeclamptic women and
controls and found a rapid decline for both markers in all groups
(Acta Obstericia et Gynecologia, 2008; 87: 146-153). However, women
showing complications of preeclampsia postpartum were not included
in the study.
[0013] Reddy et al. found that the concentration levels of sFlt-1
(and activin A, but not soluble Endoglin) increase during labour in
preeclamptic women compared to normal control women (PLoS ONE,
2009, 4(2), e4453). They found that in both groups sFlt-1 levels
decline within 24 hours. Women showing postpartum complications
were not included in the study.
[0014] WO 2013/068475 describes a method for diagnosing pregnant
women at risk for developing preeclampsia (between about week 15
and about week 34 of gestation) within a short period of time by
two measurements of the Ratio (sFlt-1/PlGF). Women are at risk if
ratio 2 to ratio is increased by a factor of at least about 3.
[0015] WO 2014/001244 describes a method for diagnosing whether a
pregnant women is not at risk for developing preeclampsia
(eclampsia and/or HELLP syndrome) within a short period of time
(1-2 weeks); the pregnant subject is between about week 20 and
about week 40 of gestation.
[0016] Prager et al. 2013 monitored the ratios of sFlt-1/PIGF in
pregnant women with onset of HELLP syndrome before delivery under
cortison therapy. Women showing postpartum HELLP syndrome were not
included in the study (Prager, R; Eckart, A; Meint, P;
Seelbach-Gobel, B: Verhalten der Angiogenesefaktoren (PlGF and
sFlt-1) unter prapartaler Dexamethason-Therapie beim HELLP-Syndrom,
Z Geburtshilfe Neonatol 2013; 217: Po01_6 DOI:
10.1055/s-0033-1361384).
[0017] Early diagnosis of postpartum complications is important
because the morbidity and mortality rates associated with these
complications that have been reported are high. For example,
postpartum preeclampsia requires prompt treatment. Left untreated,
postpartum preeclampsia can result in seizures and other serious
complications. Thus, a reliable assay for identifying a subject who
is at risk of developing postpartum HELLP syndrome, postpartum
eclampsia and postpartum preeclampsia is not yet available but
nevertheless highly desired.
SUMMARY
[0018] The technical problem underlying the present invention can
be seen as the provision of means and methods for complying with
the aforementioned needs. The technical problem is solved by the
embodiments characterized in the claims and herein below.
[0019] Advantageously, it has been found in the context of the
studies underlying the present invention that the sFlt-1/PlGF or
Endoglin/PlGF ratio in a female subject with an uneventful
pregnancy, serve as biomarker for predicting the risk of said
subject of developing a preeclampsia related adverse outcome after
delivery of baby, in particular of developing postpartum
preeclampsia, postpartum eclampsia, and/or postpartum HELLP
syndrome. Remarkably, an increase of the ratio of sFlt-1/PlGF or
Endoglin/PlGF obtained after delivery of baby as compared to a
sample obtained before delivery of baby was indicative for a risk
of developing a preeclampsia related adverse outcome after delivery
of baby, whereas a decrease of the ratio of sFlt-1/PlGF or
Endoglin/PlGF was indicative for a subject who is not at risk of
developing a preeclampsia related adverse outcome after delivery of
baby.
[0020] Thanks to the present invention, it is possible to more
reliably assess the risk of developing at least one preeclampsia
related adverse outcome after delivery of baby, based on a reliable
indicator. Moreover, the time consuming, expensive and cumbersome
diagnostic measures can be avoided when applying the method of the
invention and suitable supportive measures can be initiated. Health
care management shall greatly benefit from the method of the
present invention.
[0021] Accordingly, the present invention relates to a method for
predicting the risk of a female subject to develop at least one
preeclampsia related adverse outcome after delivery of baby (and
thus to suffer from at least one preeclampsia related adverse
outcome after delivery of baby), said method comprising the steps
of [0022] a) measuring in a first sample obtained from a female
subject with an uneventful pregnancy before delivery of baby [0023]
i) the level of the biomarker sFlt-1 (soluble fms-like tyrosine
kinase-1) or the level of the biomarker Endoglin, and [0024] ii)
the level of the biomarker PlGF (Placental Growth Factor), [0025]
b) calculating a first ratio of the levels of the biomarkers as
measured in step a), [0026] c) measuring in a second sample
obtained from said female subject after delivery of baby the levels
of the biomarkers as measured in step a), [0027] d) calculating a
second ratio of the levels measured in step c), and [0028] e)
comparing the second ratio to the first ratio.
[0029] In an embodiment, step e) of comparing the second ratio to
the first ratio is carried out by calculating a ratio of the second
ratio to the first ratio (or vice versa).
[0030] Preferably, the risk of the female subject to develop at
least one preeclampsia related adverse outcome after delivery of
baby is predicted based in the results of the comparison step
carried out in step (e). Accordingly, the aforementioned method may
further comprise the further step of predicting (or providing a
prediction of) the risk of the female subject to develop at least
one preeclampsia related adverse outcome after delivery of baby
based on the results of the comparison step.
[0031] The method of the present invention, preferably, is an ex
vivo or in vitro method. Moreover, it may comprise steps in
addition to those explicitly mentioned above. For example, further
steps may relate to sample pre-treatments or evaluation of the
results obtained by the method. The method may be carried out
manually or assisted by automation. Preferably, the measurement
steps, the calculation steps and the comparison step may in total
or in part be assisted by automation, e.g., by a suitable robotic
and sensory equipment for the measurement, a computer-implemented
calculation algorithm on a data processing device in the
calculation steps, or comparison and/or diagnosis algorithm on a
data processing device in the comparison step.
DETAILED DESCRIPTION
[0032] In accordance with the present invention, the risk of the
female subject to develop and, thus, to suffer from at least one
preeclampsia related adverse outcome after delivery of baby shall
be predicted. Preeclampsia related adverse outcomes that develop
after delivery of baby are well known by the skilled person. As
used herein, the term preferably refers to a preeclampsia related
adverse outcome which develops after pregnancy. Preferably, the at
least one preeclampsia related adverse outcome after delivery of
baby is selected from the group consisting of postpartum HELLP
syndrome, postpartum preeclampsia, and postpartum eclampsia,
postpartum cerebral hemorrhage, postpartum renal failure, in
particular postpartum acute renal failure, postpartum pulmonary
edema, in particular acute postpartum pulmonary edema, postpartum
cerebral edema, and postpartum liver rupture, disseminated
intravascular coagulation (DIC) and postpartum maternal death. More
preferably, at least one preeclampsia related adverse outcome after
delivery of baby is selected from the group consisting of
postpartum HELLP syndrome, postpartum preeclampsia, and postpartum
eclampsia. Accordingly, it is preferably predicted whether the
female subject is a risk of developing postpartum HELLP syndrome,
postpartum preeclampsia, and/or postpartum eclampsia.
[0033] The term "at least one preeclampsia related adverse outcome"
refers to one preeclampsia related adverse outcome or more than
one, i.e. two or three (or even more) preeclampsia related outcomes
(since e.g. eclampsia usually follows preeclampsia).
[0034] The term "preeclampsia" as used herein refers to a medical
condition which is characterized by hypertension and proteinuria.
Preeclampsia can occur in pregnant female subjects before and after
delivery of baby, i.e. before and after childbirth. In the context
of the present invention, the risk of a subject to suffer from
preeclampsia after delivery of a baby shall be predicted, rather
than to suffer from preeclampsia during pregnancy. Most cases of
postpartum preeclampsia develop within 48 hours after childbirth.
However, postpartum preeclampsia sometimes develops up to four to
six weeks after childbirth. This is known as late postpartum
preeclampsia. Preferably, the pregnancy-induced hypertension is
identified to be present in a subject by two blood pressure
measurements of 140 mmHg (systolic) to 90 mmHg (diastolic) or more,
wherein said two measurements have been made at least 6 hours
apart. Proteinuria is, preferably, identified to be present by 300
mg/dL protein or more, in particular, in a 24-hour urine sample.
Also preferably, proteinuria is identified by protein dipstick
analysis (if .gtoreq.2+), or if .gtoreq.30 mg/dL protein in present
in a spot urine sample, or protein/creatinine ratio is .gtoreq.30
mg protein/mmol creatinine in spot urine.
[0035] Preeclampsia may progress to eclampsia, a life-threatening
disorder characterized by the appearance of tonicclonic seizures or
coma conditions. Symptoms associated with severe preeclampsia are
oligouria of less than 500 ml within 24 hours, cerebral or visual
disturbance, pulmonary edema or cyanosis, epigastric- or right
upper quadrant-pain, impaired liver function, thrombocytopenia.
[0036] The term "HELLP syndrome" is well known in the art. HELLP
syndrome is a life-threatening obstetric complication usually
considered complication of preeclampsia. Both conditions usually
occur during the later stages of pregnancy, or after delivery of
baby. In the context of the present invention, the risk of a female
subject to suffer from HELLP syndrome after delivery shall be
predicted. The HELLP syndrome is associated with a high risk of
adverse outcomes such as renal failure, subcapsular hepatic
hematoma, recurrent preeclampsia, or even death. "HELLP" is an
abbreviation of the three main features of the syndrome: Hemolysis,
Elevated Liver enzymes, and Low Platelet count. HELLP syndrome can
be difficult to diagnose due to the variability of symptoms among
patients (frequently patients have no symptoms other than general
abdominal pain), and early diagnosis is key in reducing morbidity.
If not treated in a timely manner, patients can become critically
ill or die due to liver rupture/hemorrhage or cerebral edema. In a
patient with possible HELLP syndrome, a batch of blood tests is
performed: a full blood count, a coagulation panel, liver enzymes,
electrolytes, and renal function studies. Often, fibrin degradation
product (FDP) levels are determined, which can be elevated. Lactate
dehydrogenase is a marker of hemolysis and is elevated (.gtoreq.600
U/liter). Proteinuria is present but can be mild.
[0037] Further details of preeclampsia and the accompanying
symptoms as well as the follow up diseases such as HELLP syndrome
or eclampsia are to be found in standard text books of medicine or
Guidelines of the relevant medical societies. Details can be found,
e.g., in ACOG Practice Bulletin, Clinical Management Guidelines for
Obstetrician--Gynecologists, no.: 33, January 2002 or Haram K,
Svendsen E, Abildgaard U. The HELLP syndrome: clinical issues and
management. A review. BMC Pregnancy and Childbirth 2009; 9(8).
http://dx.doi.org/10.1186/1471-2393-9-8 or DGGG. S1-Leitlinie:
Diagnostik und Therapie hypertensiver Schwangerschaftserkrankun-gen
der Deutschen Gesellschaft fur Gynakologie und Geburtshilfe, see
citation above.
[0038] The "subject" as referred to herein is, preferably, a
mammal. Mammals include, but are not limited to, domesticated
animals (e.g., cows, sheep, cats, dogs, and horses), primates
(e.g., humans and non-human primates such as monkeys), rabbits, and
rodents (e.g., mice and rats). Preferably, the subject is a human
subject. The subject according to the present invention shall be a
female subject. The female subject shall be pregnant at the time at
which the first sample is obtained. However, the second sample
shall be obtained after delivery of baby. The terms "subject" and
"patient" may be used interchangeably herein.
[0039] Preferably, the female subject shall be a subject with an
uneventful pregnancy. The term "uneventful pregnancy" is well
understood by the skilled person. In particular, it is envisaged
that a subject with an uneventful pregnancy did not exhibit
pre-eclampsia (in particular severe preeclampsia), eclampsia,
and/or a HELLP syndrome during pregnancy (i.e. during the present
pregnancy). Accordingly, the subject with an uneventful pregnancy
preferably did not suffer from pre-eclampsia (in particular severe
preeclampsia), eclampsia, and/or the HELLP syndrome before delivery
of baby (in particular during the present pregnancy). In
particular, it is envisaged that the subject did not suffer from
pre-eclampsia (in particular severe preeclampsia), eclampsia, and
the HELLP syndrome before delivery of baby.
[0040] Thus, at the time at which the first sample is obtained, the
subject according to the present invention, preferably, shall
preferably exhibit no clinical diagnosis of preeclampsia,
eclampsia, and/or the HELLP syndrome before delivery. However, the
subject according to the present invention may exhibit at least one
symptom selected from the group consisting of epigastric pain,
headache, visual disturbance, hypertension and edema and may, thus,
suspected to be at risk of developing (and thus to suffer from) at
least one preeclampsia related adverse outcome after delivery of
baby, in particular of developing a postpartum HELLP syndrome,
postpartum preeclampsia, and/or postpartum eclampsia. In an
embodiment, the subject exhibits said at least one symptom shortly
before delivery of baby, in particular the subject exhibits said at
least one symptom at the time at which the first sample is
obtained.
[0041] Further, it is envisaged that the subject with an uneventful
pregnancy suffered from mild preeclampsia before delivery baby,
i.e. in the present pregnancy. In this case, the risk refers to
developing at least one severe preeclampsia related adverse outcome
after delivery of baby. Preferably, the severe preeclampsia related
adverse outcome is selected from postpartum HELLP syndrome,
postpartum eclampsia and postpartum severe preeclampsia. The terms
"mild preeclampsia" and "severe preeclampsia" are well known in the
art. The term "mild preeclampsia" preferably refers to the of
hypertension (in particular of a blood pressure .gtoreq.140/90 mm
Hg) on 2 occasions, at least 6 hours apart, but without evidence of
end-organ damage, in a woman who was normotensive before 20 weeks'
gestation. The term "severe preeclampsia" refers to preeclampsia
with at least one of the following symptoms, systolic blood
pressure of 160 mm Hg or higher or diastolic blood pressure of 110
mm Hg or higher on 2 occasions at least 6 hours apart, proteinuria
of more than 5 g in a 24-hour collection or more than 3+ on 2
random urine samples collected at least 4 hours apart, Oliguria
(<400 mL in 24 hours), persistent headaches, epigastric pain
and/or impaired liver function and thrombocytopenia. For a
definition of the terms, see e.g. Sibai et al. Lancet. 2005 Feb.
26-Mar. 4; 365(9461):785-99. which herewith is incorporated by
reference with respect to its entire disclosure content.
[0042] Also preferably, the subject may be a risk person for
developing at least one preeclampsia related adverse outcome after
delivery of baby, in particular of postpartum HELLP syndrome,
postpartum preeclampsia, and/or postpartum eclampsia. A risk person
preferably is a female subject being older than 40 years and/or a
female subject in the first pregnancy, have a family history of
pre-eclampsia (e.g., pre-eclampsia in a mother or sister), have a
prior history of pre-eclampsia in previous pregnancy or after
delivery of a previous baby, have a body mass index at or above 35
kg/m.sup.2 at first contact, have a multiple pregnancy or
pre-existing vascular disease such as hypertension or diabetes,
e.g. as described in the NICE (National Institute for Health and
Care Excellence)) Antenatal Care guideline CG62, March 2008.
[0043] The delivery technique may be any technique deemed
appropriate. Preferably, the delivery technique includes one of a
non-induced vaginal birth, a cesarean section, and a drug-induced
labor. In preferred embodiment, a single baby is delivered.
However, it is also envisaged that more than one baby is delivered.
Preferably, the baby is apparently healthy after delivery.
[0044] In accordance with the method of the present invention, the
risk of female subject to develop at least one preeclampsia related
adverse outcome after delivery of baby, in particular postpartum
HELLP syndrome, postpartum preeclampsia, and/or postpartum
eclampsia shall be predicted, and, thus, the risk of a said subject
to suffer from said adverse outcome. Preferably, it is predicted
whether said adverse outcome develops immediately after delivery of
baby. The term "immediately after delivery of baby" in connection
with said adverse outcome, in particular with postpartum HELLP
syndrome, postpartum preeclampsia, and/or postpartum eclampsia is
well understood by the skilled person. Preferably, the risk is
predicted to develop at least one preeclampsia related adverse
outcome within two weeks, more preferably within seven days, even
more preferably within 72 hours, or, most preferably, within 48
hours after delivery of baby. Preferably, the subject does not
suffer from at least one preeclampsia related adverse outcome, in
particular postpartum HELLP syndrome, postpartum preeclampsia
and/or postpartum eclampsia at the time at which the second sample
is obtained.
[0045] The term "predicting the risk" as used herein, preferably,
refers to assessing the probability according to which at least one
preeclampsia related adverse outcome will develop in a subject
after delivery of baby or not. More preferably, the
risk/probability of developing (and thus of suffering from) at
least one preeclampsia related adverse outcome within a certain
time window after delivery of baby is predicted. As set forth
above, the predictive window, preferably, is an interval at two
weeks, at seven days, of 72 hours, 48 hours, or any intermitting
time range after delivery of baby. In a particular preferred
embodiment of the present invention, the predictive window,
preferably, is an interval of 48 hours. Preferably, said the
predictive window is calculated from the delivery of baby. Also
preferably, said predictive window is calculated from the time
point at which the second sample has been obtained.
[0046] As will be understood by those skilled in the art, such a
prediction is usually not intended to be correct for 100% of the
subjects. The term, however, requires that prediction can be made
for a statistically significant portion of subjects in a proper and
correct manner. Whether a portion is statistically significant can
be determined without further ado by the person skilled in the art
using various well known statistic evaluation tools, e.g.,
determination of confidence intervals, p-value determination,
Student's t-test, Mann-Whitney test etc. Details are found in Dowdy
and Wearden, Statistics for Research, John Wiley & Sons, New
York 1983. Preferred confidence intervals are at least 90%, at
least 95%, at least 97%, at least 98%, or at least 99%. The
p-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001.
Preferably, the probability envisaged by the present invention
allows that the prediction of an increased, normal or decreased
risk will be correct for at least 60%, at least 70%, at least 80%,
or at least 90% of the subjects of a given cohort or population.
The term, preferably, relates to predicting whether a subject is at
elevated risk or reduced risk as compared to the average risk for
developing at least one preeclampsia related adverse outcome after
delivery of baby in a population of female subjects immediately
after delivery of baby.
[0047] The term "predicting the risk of developing at least one
preeclampsia related adverse outcome after delivery of baby" as
used herein means that the subject to be analyzed by the method of
the present invention is allocated either into the group of
subjects being at risk of developing said at least one adverse
outcome, or into the group of subjects being not at risk of
developing at least one preeclampsia related adverse outcome. A
risk of developing said at least one adverse outcome as referred to
in accordance with the present invention, preferably, means that
the risk of developing said at least one adverse outcome is
elevated (within the predictive window). Preferably, said risk is
elevated as compared to the average risk in a cohort of female
subjects immediately after delivery of baby (i.e. a group of such
subjects). If a subject is not at risk of developing said a
preeclampsia related adverse outcome as referred to in accordance
with the present invention, preferably, the risk of developing said
adverse outcome shall be reduced (within the predictive window).
Preferably, said risk is reduced as compared to the average risk in
a cohort of female subjects immediately after delivery of baby. A
subject who is at risk of developing said at least one adverse
outcome preferably has a risk of 80% or larger, or, more preferably
of 60% or larger of developing said at least one adverse outcome,
preferably, immediately after delivery of baby. A subject who is at
not at risk of developing a preeclampsia related adverse outcome
preferably has a risk of lower than 20%, more preferably of lower
than, 10% or lower, or more preferably of 5% or lower of developing
said at least one adverse outcome, preferably, immediately after
delivery of baby.
[0048] In accordance with the present invention, a risk prediction
may be provided. The phrase "providing a prediction" as used herein
refers to using the information or data generated relating to first
and second ratio in a sample of a patient to predict the risk of
the subject to develop at least one preeclampsia related adverse
outcome after delivery of baby. The information or data may be in
any form, written, oral or electronic. In some embodiments, using
the information or data generated includes communicating,
presenting, reporting, storing, sending, transferring, supplying,
transmitting, dispensing, or combinations thereof. In some
embodiments, communicating, presenting, reporting, storing,
sending, transferring, supplying, transmitting, dispensing, or
combinations thereof are performed by a computing device, analyzer
unit or combination thereof. In some further embodiments,
communicating, presenting, reporting, storing, sending,
transferring, supplying, transmitting, dispensing, or combinations
thereof are performed by a laboratory or medical professional.
[0049] The term "sample" refers to a sample of a body fluid, to a
sample of separated cells or to a sample from a tissue or an organ.
Samples of body fluids can be obtained by well-known techniques and
include, samples of blood, plasma, serum, urine, lymphatic fluid,
sputum, ascites, or any other bodily secretion or derivative
thereof. Tissue or organ samples may be obtained from any tissue or
organ by, e.g., biopsy. Separated cells may be obtained from the
body fluids or the tissues or organs by separating techniques such
as centrifugation or cell sorting. E.g., cell-, tissue- or organ
samples may be obtained from those cells, tissues or organs which
express or produce the biomarker. The sample may be frozen, fresh,
fixed (e.g. formalin fixed), centrifuged, and/or embedded (e.g.
paraffin embedded), etc. The cell sample can, of course, be
subjected to a variety of well-known post-collection preparative
and storage techniques (e.g., nucleic acid and/or protein
extraction, fixation, storage, freezing, ultrafiltration,
concentration, evaporation, centrifugation, etc.) prior to
assessing the level of the marker in the sample. Likewise, biopsies
may also be subjected to post-collection preparative and storage
techniques, e.g., fixation.
[0050] In an embodiment the sample is a blood, plasma or, in
particular, a serum sample. Preferably, the sample is a venous
blood, venous serum or venous plasma sample derived from the female
subject. Also preferably, the sample is a urine sample.
[0051] In accordance with the present invention, it is envisaged to
measure the level of a biomarker as referred to herein in a first
and a second sample from the female subject. The first sample shall
have been obtained from the female subject before delivery of baby,
in particular immediately before delivery of baby. Thus, the first
sample, preferably, shall have been obtained within two weeks or
one week, more preferably, within three days, even more preferably
within 48 hours, or most preferably within 24 hours before delivery
of baby. Further, it is envisaged to obtain the first sample within
12 hours before delivery of baby.
[0052] The "second sample" is, preferably, understood as a sample
which is obtained in order to reflect a change of the second ratio
as compared to the first ratio in the first sample. The second
sample shall be obtained after the first sample. In particular, the
second sample shall be obtained after delivery of baby. Preferably,
the second sample has been obtained within 72 hours or within 48
hours after delivery of baby, more preferably, within 24 hours
after delivery of baby, even more preferably within 16 hours, and
most preferably within 12 hours after delivery of baby.
[0053] Preferably, the second sample is not obtained too early
after the first sample (in order to observe a sufficiently
significant change to allow the risk prediction). Thus, the "second
sample" is preferably obtained not earlier than 10 hours, more
preferably, not earlier than 8 hours, or most preferably not
earlier than 6 hours after the first sample. Thus, there should be
an interval of preferably at least 10 hours, more preferably, of at
least 8 hours and most preferably at least 6 hours between
obtaining the first and the second sample.
[0054] Also preferably, it is envisaged that the first sample is
obtained not earlier than three hours before delivery of baby and
that the second sample is obtained not earlier than three hours
after delivery of baby. Also, the first sample may be obtained not
earlier than five hours before delivery of baby and the second
sample may be obtained not earlier than five hours after delivery
of baby.
[0055] The term "delivery" in connection with childbirth is well
understood by the skilled person. It is the culmination of a period
of pregnancy with the expulsion of one or more newborn infants from
a woman's uterus. As used herein, the expression "delivery of baby"
preferably refers to the birth of the baby. More preferably, the
delivery of baby is the time point at which the fetus is expelled
from the subject's uterus. Most preferably, the delivery of baby is
the time point at which the baby starts breathing. It is also
envisaged that the delivery of baby is the time point at which the
placenta is delivered.
[0056] In an embodiment of the present invention, there are no
maternal or fetal complications during childbirth.
[0057] It is to be understood that the first and the second sample
are the same kind of sample. E.g., if the first sample is a serum
sample the second sample shall be a serum sample as well.
[0058] The term "measuring" the level of a marker as referred to
herein refers to the quantification of the biomarker, e.g. to
determining the level of the biomarker in the sample, employing
appropriate methods of detection described elsewhere herein.
[0059] In an embodiment, the level of the at least one biomarker is
measured by contacting the sample with a detection agent that
specifically binds to the respective marker, thereby forming a
complex between the agent and said marker, detecting the level of
complex formed, and thereby measuring the level of said marker.
[0060] The biomarkers as referred to herein can be detected using
methods generally known in the art. Methods of detection generally
encompass methods to quantify the level of a biomarker in the
sample (quantitative method). It is generally known to the skilled
artisan which of the following methods are suitable for qualitative
and/or for quantitative detection of a biomarker. Samples can be
conveniently assayed for, e.g., proteins using Westerns and
immunoassays, like ELISAs, RIAs, fluorescence-based immunoassays,
which are commercially available. Further suitable methods to
detect biomarker include measuring a physical or chemical property
specific for the peptide or polypeptide such as its precise
molecular mass or NMR spectrum. Said methods comprise, e.g.,
biosensors, optical devices coupled to immunoassays, biochips,
analytical devices such as mass-spectrometers, NMR-analyzers, or
chromatography devices. Further, methods include microplate
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).
[0061] For the detection of biomarker proteins as referred to
herein a wide range of immunoassay techniques using such an assay
format are available, see, e.g., U.S. Pat. Nos. 4,016,043,
4,424,279, and 4,018,653. These include both single-site and
two-site or "sandwich" assays of the noncompetitive types, as well
as in the traditional competitive binding assays. These assays also
include direct binding of a labeled antibody to a target
biomarker.
[0062] Sandwich assays are among the most useful and commonly used
immunoassays.
[0063] Methods for measuring electrochemiluminescent phenomena are
well-known. Such methods make use of the ability of special metal
complexes to achieve, by means of oxidation, an excited state from
which they decay to ground state, emitting
electrochemiluminescence. For review see Richter, M. M., Chem. Rev.
104 (2004) 3003-3036.
[0064] Biomarkers can also be detected by generally known methods
including magnetic resonance spectroscopy (NMR spectroscopy), Gas
chromatography-mass spectrometry (GC-MS), Liquid
chromatography-mass spectrometry (LC-MS), High and ultra-HPLC HPLC
such as reverse phase HPLC, for example, ion-pairing HPLC with dual
UV-wavelength detection, capillary electrophoresis with
laser-induced fluorescence detection, anion exchange chromatography
and fluorescent detection, thin layer chromatography.
[0065] Preferably, measuring the level of a biomarker as referred
to herein comprises the steps of (a) contacting a cell capable of
eliciting a cellular response the intensity of which is indicative
of the level of the peptide or polypeptide with the said peptide or
polypeptide for an adequate period of time, (b) measuring the
cellular response. For measuring cellular responses, the sample or
processed sample is, preferably, added to a cell culture and an
internal or external cellular response is measured. The cellular
response may include the measurable expression of a reporter gene
or the secretion of a substance, e.g. a peptide, polypeptide, or a
small molecule. The expression or substance shall generate an
intensity signal which correlates to the level of the peptide or
polypeptide.
[0066] Also preferably, measuring the level of a peptide or
polypeptide comprises 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.
[0067] Measuring the level of a peptide or polypeptide may,
preferably, comprises the steps of (a) contacting the peptide with
a specific binding agent, (b) (optionally) removing non-bound
binding agent, (c) measuring the level of bound binding agent, i.e.
the complex of the binding agent formed in step(a). According to a
preferred embodiment, said steps of contacting, removing and
measuring may be performed by an analyzer unit of the system
disclosed herein. According to some embodiments, said steps may be
performed by a single analyzer unit of said system or by more than
one analyzer unit in operable communication with each other. For
example, according to a specific embodiment, said system disclosed
herein may include a first analyzer unit for performing said steps
of contacting and removing and a second analyzer unit, operably
connected to said first analyzer unit by a transport unit (for
example, a robotic arm), which performs said step of measuring.
[0068] The bound binding agent, i.e. the binding agent or the
binding agent/peptide complex, will generate an intensity signal.
Binding according to the present invention includes both covalent
and non-covalent binding. A binding agent according to the present
invention can be any compound, e.g., a peptide, polypeptide,
nucleic acid, or small molecule, binding to the peptide or
polypeptide described herein. Preferred binding agents 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, e.g. nucleic acid or peptide aptamers. Methods to
prepare such binding agents are well-known in the art. For example,
identification and production of suitable antibodies or aptamers is
also offered by commercial suppliers. The person skilled in the art
is familiar with methods to develop derivatives of such binding
agents 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)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 can be prepared by several methods
well known in the art. Preferably, the binding agent 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. Preferably, the specifically bound peptide or polypeptide
should be bound with at least 3 times higher, more preferably at
least 10 times higher and even more preferably at least 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, e.g. according to its
size on a Western Blot, or by its relatively higher abundance in
the sample. Binding of the binding agent can be measured by any
method known in the art. Preferably, said method is
semi-quantitative or quantitative. Further suitable techniques for
the determination of a polypeptide or peptide are described in the
following.
[0069] Binding of a binding agent may be measured directly, e.g. by
NMR or surface plasmon resonance. Measurement of the binding of a
binding agent, according to preferred embodiments, is performed by
an analyzer unit of a system disclosed herein. Thereafter, a level
of the measured binding may be calculated by a computing device of
a system disclosed herein. If the binding agent also 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
level of a protease can be measured by measuring the level of
cleaved substrate, e.g. on a Western Blot). Alternatively, the
binding agent may exhibit enzymatic properties itself and the
"binding agent/peptide or polypeptide" complex or the binding agent
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 measurement of enzymatic
reaction products, preferably the level of substrate is saturating.
The substrate may also be labeled with a detectable label prior to
the reaction. Preferably, the sample is contacted with the
substrate for an adequate period of time. An adequate period of
time refers to the time necessary for a detectable, preferably
measurable, level of product to be produced. Instead of measuring
the level of product, the time necessary for appearance of a given
(e.g. detectable) level of product can be measured. Third, the
binding agent may be coupled covalently or non-covalently to a
label allowing detection and measurement of the binding agent.
Labeling may be done by direct or indirect methods. Direct labeling
involves coupling of the label directly (covalently or
non-covalently) to the binding agent. Indirect labeling involves
binding (covalently or non-covalently) of a secondary binding agent
to the first binding agent. The secondary binding agent should
specifically bind to the first binding agent. Said secondary
binding agent may be coupled with a suitable label and/or be the
target (receptor) of tertiary binding agent binding to the
secondary binding agent. The use of secondary, tertiary or even
higher order binding agents is often used to increase the signal.
Suitable secondary and higher order binding agents may include
antibodies, secondary antibodies, and the well-known
streptavidin-biotin system (Vector Laboratories, Inc.). The binding
agent 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
binding agents. Suitable tags include biotin, digoxygenin, His-Tag,
Glutathion-S-Transferase, FLAG, GFP, myctag, influenza A virus
hae-magglutinin (HA), maltose binding protein, and the like. In the
case of a peptide or polypeptide, the tag is preferably at the
N-terminus and/or C-terminus. Suitable labels are any labels
detectable by an appropriate detection method. Typical 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 include e.g. 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-BLIP
(4-nitro blue tetrazolium chloride and
5-bromo-4-chloro-3-indolyl-phosphate, available as ready-made stock
solution from Roche Diagnostics), CDP-Star.TM. (Amersham
Bio-sciences), ECF.TM. (Amersham Biosciences). A suitable
enzyme-substrate combination may result in a colored reaction
product, fluorescence or chemoluminescence, which can be measured
according to methods known in the art (e.g. using a light-sensitive
film or a suit-able camera system). As for measuring the enzymatic
reaction, the criteria given above apply analogously. Typical
fluorescent labels include fluorescent proteins (such as GFP and
its derivatives), Cy3, Cy5, Texas Red, Fluorescein, and the Alexa
dyes (e.g. Alexa 568). Further fluorescent labels are available
e.g. from Molecular Probes (Oregon). Also the use of quantum dots
as fluorescent labels is contemplated. A radioactive label can be
detected by any method known and appropriate, e.g. a
light-sensitive film or a phosphor imager.
[0070] The level of a peptide or polypeptide may be, also
preferably, determined as follows: (a) contacting a solid support
comprising a binding agent for the peptide or polypeptide as
specified above with a sample comprising the peptide or polypeptide
and (b) measuring the level peptide or polypeptide which is bound
to the support. The binding agent, preferably chosen from the group
consisting of nucleic acids, peptides, polypeptides, antibodies and
aptamers, is preferably present on a solid support in immobilized
form. Materials for manufacturing solid supports are well known in
the art and include, inter alia, 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 binding agent 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 can be either
soluble or insoluble for the purposes of the invention. Suitable
methods for fixing/immobilizing said binding agent are well 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 invention
(No-lan 2002, Trends Biotechnol. 20(1):9-12). In such suspension
arrays, the carrier, e.g. a microbead or microsphere, is present in
suspension. The array consists of different microbeads or
microspheres, possibly labeled, carrying different binding agents.
Methods of producing such arrays, for example based on solid-phase
chemistry and photo-labile protective groups, are generally known
(U.S. Pat. No. 5,744,305).
[0071] In an embodiment of the present invention, the levels of the
biomarkers as referred to herein are measured by using the assays
described in the Examples section.
[0072] In another embodiment of the method of the present
invention, the measurement in step a) (or in steps a) and c)) may
be carried out by an analyzer unit, in particular by an analyzer
unit as defined elsewhere herein.
[0073] The term "binding agent" refers to a molecule that comprises
a binding moiety which specifically binds the corresponding to the
respective biomarker. Examples of "binding agent" are a aptamer,
antibody, antibody fragment, peptide, peptide nucleic acid (PNA) or
chemical compound.
[0074] The term "specific binding" or "specifically bind" refers to
a binding reaction wherein binding pair molecules exhibit a binding
to each other under conditions where they do not significantly bind
to other molecules. The term "specific binding" or "specifically
binds", when referring to a protein or peptide as biomarker, refers
to a binding reaction wherein a binding agent binds to the
corresponding biomarker with an affinity of at least 10.sup.-7 M.
The term "specific binding" or "specifically binds" preferably
refers to an affinity of at least 10.sup.-8 M or even more
preferred of at least 10.sup.-9 M for its target molecule. The term
"specific" or "specifically" is used to indicate that other
molecules present in the sample do not significantly bind to the
binding agent specific for the target molecule. Preferably, the
level of binding to a molecule other than the target molecule
results in a binding affinity which is only 10% or less, more
preferably only 5% or less of the affinity to the target
molecule.
[0075] Examples of "binding agents" or "agents" are a nucleic acid
probe, nucleic acid primer, DNA molecule, RNA molecule, aptamer,
antibody, antibody fragment, peptide, peptide nucleic acid (PNA) or
chemical compound. A preferred agent is an antibody which
specifically binds to the biomarker to be measured. The term
"antibody" herein is used in the broadest sense and encompasses
various antibody structures, including but not limited to
monoclonal antibodies, polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they exhibit the desired antigen-binding activity.
Preferably, the antibody is a polyclonal antibody. More preferably,
the antibody is a monoclonal antibody.
[0076] Another binding agent that can be applied, in an aspect, may
be an aptamere which specifically binds to the at least one marker
in the sample. The term "specific binding" or "specifically binds",
when referring to a nucleic acid aptamer as a binding agent, refers
to a binding reaction wherein a nucleic acid aptamer binds to the
corresponding target molecule with an affinity in the low nM to pM
range.
[0077] In yet an aspect the, sample is removed from the complex
formed between the binding agent and the at least one marker prior
to the measurement of the level of formed complex. Accordingly, in
an aspect, the binding agent may be immobilized on a solid support.
In yet an aspect, the sample can be removed from the formed complex
on the solid support by applying a washing solution. The formed
complex shall be proportional to the level of the at least one
marker present in the sample. It will be understood that the
specificity and/or sensitivity of the binding agent to be applied
defines the degree of proportion of at least one marker comprised
in the sample which is capable of being specifically bound. Further
details on how the determination can be carried out are also found
elsewhere herein. The level of formed complex shall be transformed
into a level of at least one marker reflecting the level indeed
present in the sample. Such a level, in an aspect, may be
essentially the level present in the sample or may be, in another
aspect, an level which is a certain proportion thereof due to the
relationship between the formed complex and the level present in
the original sample.
[0078] The term "sFlt-1" as used herein refers to a polypeptide
which is a soluble form of the fms-like tyrosine kinase 1. The
polypeptide is also referred to as soluble VEGF receptor 1 (sVEGF
R1) in the art (see, e.g., Sunderji 2010, Am J Obstet Gynecol 202:
40e1-7). It was identified in conditioned culture medium of human
umbilical vein endothelial cells. The endogenous sFlt-1 receptor is
chromatographically and immunologically similar to recombinant
human sFlt-1 and binds [125I] VEGF with a comparable high affinity.
Human sFlt-1 is shown to form a VEGF-stabilized complex with the
extracellular domain of KDR/Flk-1 in vitro. Preferably, sFlt-1
refers to human sFlt-1 as describe in Kendall 1996, Biochem Biophs
Res Commun 226(2): 324-328; for amino acid sequences, see, e.g.,
also Genebank accession numbers P17948, GI: 125361 for human and
BAA24499.1, GI: 2809071 for mouse sFlt-1 (Genbank is available from
the NCBI, USA under www.ncbi.nlm.nih.gov/entrez). The term also
encompasses variants of the aforementioned human sFlt-1
polypeptides. Such variants have at least the same essential
biological and immunological properties as the aforementioned
sFlt-1 polypeptide. In particular, they share the same essential
biological and immunological properties 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 sFlt-1 polypeptides. Moreover, it
is to be understood that a variant as referred to in accordance
with the present invention 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, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%,
97%, 98%, or 99% identical with the amino sequence of the specific
sFlt-1 polypeptide, preferably over the entire length of the human
sFlt-1, respectively. The degree of identity between two amino acid
sequences can be determined by algorithms well known in the art.
Preferably, the degree of identity is to 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 is
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
disclosed by Smith 1981, Add. APL. Math. 2:482, by the homology
alignment algorithm of Needleman 1970, J. Mol. Biol. 48:443, by the
search for similarity method of Pearson 1988, Proc. Natl. Acad Sci.
(USA) 85: 2444, by computerized implementations of these algorithms
(GAP, BESTFIT, BLAST, FAST, 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
are preferably 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 are used.
Variants referred to above may be allelic variants or any other
species specific homologs, paralogs, or orthologs. 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
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 sFlt-1 polypeptides. Variants are
deemed to share the same essential biological and immunological
properties 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 sFlt-1 polypeptides. A preferred assay is described in the
accompanying Examples. Further included are variants which differ
due to posttranslational modifications such as phosphorylation or
myristylation. sFlt-1 may be detected in bound or free form or as
total sFlt-1 level in a sample.
[0079] The term "Endoglin" as used herein refers to a polypeptide
having a molecular weight of 180 kDa non-reduced, 95 kDa after
reduction and 66 kDa in its reduced and N-deglycosylated form.
Preferably, the term "Endoglin" refers to soluble Endoglin. The
polypeptide is capable of forming dimers and binds to TGF-.beta.
and TGF-.beta. receptors. Preferably, Endoglin refers to human
Endoglin. More preferably, human Endoglin has an amino acid
sequence as shown in Genebank accession number AAC63386.1, GI:
3201489. Two Endoglin isoforms, S-Endoglin and L-Endoglin have been
described. L-Endoglin consists of total of 633 amino acids with a
cytoplasmic tail of 47 amino acids while S-Endoglin consists of 600
amino acids with a cytoplasmic tail of 14 amino acids. Preferably,
Endoglin as used herein is soluble Endoglin. Soluble Endoglin as
referred to herein is preferably described in EP 1 804 836 B 1.
Moreover, it is to be understood that a variant as referred to in
accordance with the present invention may 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, preferably, at least 50%, 60%, 70%, 80%, 85%,
90%, 92%, 95%, 97%, 98%, or 99% identical with the amino sequence
of the specific Endoglin. Variants may be allelic variants, splice
variants or any other species specific homologs, paralogs, or
orthologs. Moreover, the variants referred to herein include
fragments of the specific Endoglin 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 Endoglin. Variants
are deemed to share the same essential biological and immunological
properties 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 Endoglin polypeptides. A preferred assay is described in the
accompanying Examples. Further included are variants which differ
due to posttranslational modifications such as phosphorylation or
myristylation. Endoglin may be detected in bound or free form or as
total Endoglin level in a sample.
[0080] The term "PlGF (Placental Growth Factor)" as used herein,
preferably, refers to a placenta-derived growth factor which is a
polypeptide having 149 amino acids in length and being highly
homologous to the platelet-derived growth factor-like region of
human vascular endothelial growth factor (VEGF). Like VEGF, PlGF
has angiogenic activity in vitro and in vivo. For example,
biochemical and functional characterization of PlGF derived from
transfected COS-1 cells revealed that it is a glycosylated dimeric
secreted protein which is able to stimulate endothelial cell growth
in vitro (Maqlione 1993, Oncogene 8(4):925-31). Preferably, PlGF
refers to human PlGF, more preferably, to human PlGF having an
amino acid sequence as shown in Genebank accession number P49763,
GI: 17380553. The term encompasses variants of said specific human
PlGF. Such variants have at least the same essential biological and
immunological properties as the specific PlGF polypeptide. Variants
are deemed to share the same essential biological and immunological
properties 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 PlGF polypeptides. A preferred assay is described in the
accompanying Examples. Moreover, it is to be understood that a
variant as referred to in accordance with the present invention
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, preferably, at least 50%,
60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with
the amino sequence of the specific PlGF polypeptides. The degree of
identity between two amino acid sequences can be determined by
algorithms well known in the art and described elsewhere herein.
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 of the specific PlGF
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 or splice variants of the PlGF polypeptides.
Further included are variants which differ due to posttranslational
modifications such as phosphorylation or myristylation. PlGF may be
detected in bound or free form or as total PlGF level in a
sample.
[0081] The term "level" as used herein encompasses the absolute
amount of a biomarker as referred to herein, the relative amount or
concentration of the said biomarker as well as 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, e.g., 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, e.g., response amounts determined
from biological read out systems in response to the peptides or
intensity signals obtained from specifically bound ligands. It is
to be understood that values correlating to the aforementioned
amounts or parameters can also be obtained by all standard
mathematical operations.
[0082] The term "comparing" as used herein refers to comparing the
ratio of the levels of the biomarkers as referred to herein (first
ratio) in the first sample from the subject with the ratio of the
levels of said biomarkers (second ratio) in the second sample from
the subject. It is to be understood that comparing as used herein
usually refers to a comparison of corresponding parameters or
values, e.g., an absolute amount is compared to an absolute
reference amount while a concentration is compared to a reference
concentration or an intensity signal obtained from the biomarker in
a sample is compared to the same type of intensity signal obtained
from a reference sample. The comparison may be carried out manually
or computer assisted. Thus, the comparison may be carried out by a
computing device (e.g., of a system disclosed herein). The value of
the (first) ratio in the first sample from the subject and the
value of the (second) ratio in the second sample can be, e.g.,
compared to each other and the said comparison can be automatically
carried out by a computer program executing an algorithm for the
comparison. The computer program carrying out the said evaluation
will provide the desired assessment in a suitable output format.
For a computer assisted comparison, the value of the determined
ratio in the second sample may be compared to a value of the ratio
in the first sample which is 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. For a computer assisted comparison,
the value of the determined ratio in the second sample may be
compared to the value of the ratio in the first sample which is
stored in a database by a computer program. The computer program
may further evaluate the result of the comparison, i.e.
automatically provides the desired assessment in a suitable output
format.
[0083] The term "calculating a first ratio" or "calculating a
second ratio" as referred to herein relates to calculating a ratio
of the level of sFlt-1 or Endoglin and the level of PlGF by
dividing the said level or by carrying out any other comparable
mathematical calculation which puts into a relation the level of
sFlt-1 or Endoglin towards the level of PlGF. Preferably, the level
of sFlt-1 or Endoglin is divided by the level of PlGF in order to
calculate the ratio (thus, the ratio of the level of sFlt-1 or
Endoglin to the level of PlGF is calculated). Also preferably, the
level of PlGF is divided by the level of sFlt-1 or Endoglin in
order to calculate the ratio (thus, the ratio of the level of PlGF
to the level of sFlt-1 or Endoglin is calculated). The calculation
is carried out for the respective levels determined in the said
first and the said second sample separately yielding the first and
the second ratio, respectively. The calculations may be carried at
the same time, or at different times.
[0084] If the method comprises the comparison of the second ratio
to the first ratio, preferably, the following applies:
[0085] In an embodiment, the first and the second ratio are the
ratios of sFlt-1 to PlGF, or of Endoglin to PlGF. Preferably, an
increase of the second ratio (or an essentially unchanged second
ratio) as compared to the first ratio is indicative for a subject
who is at risk of developing at least one preeclampsia related
adverse outcome after delivery of baby, and/or a decrease of the
second ratio as compared to the first ratio is indicative for a
subject who is not at risk of developing a preeclampsia related
adverse outcome after delivery of baby. Also preferably, the
subject is at risk of developing at least one preeclampsia related
adverse outcome after delivery of baby, if the second ratio is
increased as compared to the first ratio, or if the second ratio is
essentially the same as the first ratio, whereas the subject is not
at risk of developing a preeclampsia related adverse outcome after
delivery of baby, if the second ratio is decreased as compared to
the first ratio.
[0086] In another embodiment, the first and the second ratio are
the ratios of PlGF to sFlt-1, or of PlGF to Endoglin. Preferably, a
decrease of the second ratio (or an essentially unchanged second
ratio) as compared to the first ratio is indicative for a subject
who is at risk of developing at least one preeclampsia related
adverse outcome after delivery of baby, and/or an increase of the
second ratio as compared to the first ratio is indicative for a
subject who is not at risk of developing a preeclampsia related
adverse outcome after delivery of baby. Also preferably, the
subject is at risk of developing at least one preeclampsia related
adverse outcome after delivery of baby, if the second ratio is
decreased as compared to the first ratio, or if the second ratio is
essentially the same as the first ratio, whereas the subject is not
at risk of developing a preeclampsia related adverse outcome after
delivery of baby, if the second ratio is increased as compared to
the first ratio.
[0087] The term "essentially unchanged" is well known in the art
and understood by the skilled person who is experienced in the
field of diagnostics. The term refers to minor changes of the
second ratio as compared to the first ratio, e.g. of less than 3 or
7%. In an embodiment the term refers to an unchanged ratio.
[0088] If step e) of comparing the second ratio to the first ratio
is carried out by calculating a ratio of the second ratio to the
first ratio (or vice versa), preferably the following applies:
[0089] If first and the second ratio are the ratios of sFlt-1 to
PlGF, or of Endoglin to PlGF, the following applies: Preferably, a
ratio which is equal or larger than 1 is indicative for a subject
who is at risk of developing at least one preeclampsia related
adverse outcome after delivery of baby, whereas a ratio which lower
than 1 is indicative for a subject who is not at risk of developing
a preeclampsia related adverse outcome after delivery of baby.
[0090] If first and the second ratio are the ratios of PlGF to
sFlt-1, or of PlGF to Endoglin the following applies: Preferably, a
ratio which is equal or lower than 1 is indicative for a subject
who is at risk of developing at least one preeclampsia related
adverse outcome after delivery of baby, whereas a ratio which
larger than 1 is indicative for a subject who is not at risk of
developing a preeclampsia related adverse outcome after delivery of
baby.
[0091] In accordance with the present invention, the terms
"increase" and "decrease" preferably refer to a statistically
significant increase and decrease respectively. Particularly, a
statistically significant increase (or decrease) is an increase (or
decrease) of a size which is considered to be statistically
significant for the risk prediction. The terms "significant" and
"statistically significant" are known to the person skilled in the
art. Whether a increase or decrease is statistically significant
can be determined without further ado by the person skilled in the
art using various well known statistic evaluation tools including
those referred to herein.
[0092] A preferred increase of the second ratio of sFlt-1 or
Endoglin to PlGF in the second sample as compared to the first
ratio of sFlt-1 or Endoglin to PlGF in the first sample which have
been found in the course of the invention to be indicative for a
subject who is at risk of developing at least one preeclampsia
related adverse outcome after delivery of baby, preferably, is an
increase of at least 3% more preferably of at least 10% and even,
more preferably, of at least 20%, and most preferably of at least
30%.
[0093] A preferred decrease of the second ratio of sFlt-1 or
Endoglin to PlGF in the second sample as compared to the first
ratio of sFlt-1 or Endoglin to PlGF in the first sample which have
been found in the course of the invention to be indicative for a
subject who is not at risk of developing a preeclampsia related
adverse outcome after delivery of baby, preferably, is decrease of
at least 10% more preferably of at least 20% and even, more
preferably, of at least 30%, and most preferably of at least
40%.
[0094] It is to be understood that the definitions and explanations
of the terms made above and below apply accordingly for all
embodiments described in this specification and the accompanying
claims.
[0095] The present invention further relates to a method for
differentiating between a subject being at risk of developing at
least one preeclampsia related adverse outcome after delivery of
baby and a subject being not at risk of developing a preeclampsia
related adverse outcome after delivery of baby, said method
comprising the steps of [0096] a) measuring in a first sample
obtained from a female subject with an uneventful pregnancy before
delivery of baby [0097] i) the level of the biomarker sFlt-1
(soluble fms-like tyrosine kinase-1) or the level of the biomarker
Endoglin, and [0098] ii) the level of the biomarker PlGF (Placental
Growth Factor), [0099] b) calculating a first ratio of the levels
of the biomarkers as measured in step a), [0100] c) measuring in a
second sample obtained from said female subject after delivery of
baby the levels of the biomarkers as measured in step a), [0101] d)
calculating a second ratio of the levels measured in step c), and
[0102] e) comparing the second ratio to the first ratio.
[0103] In an embodiment, step e) of comparing the second ratio to
the first ratio is carried out by calculating a ratio of the second
ratio to the first ratio (or vice versa).
[0104] The present invention further relates to a method for
identifying a subject being at risk of developing at least one
preeclampsia related adverse outcome after delivery of baby, said
method comprising the steps of [0105] a) measuring in a first
sample obtained from a female subject with an uneventful pregnancy
before delivery of baby [0106] i) the level of the biomarker sFlt-1
(soluble fms-like tyrosine kinase-1) or the level of the biomarker
Endoglin, and [0107] ii) the level of the biomarker PlGF (Placental
Growth Factor), [0108] b) calculating a first ratio of the levels
of the biomarkers as measured in step a), [0109] c) measuring in a
second sample obtained from said female subject after delivery of
baby the levels of the biomarkers as measured in step a), [0110] d)
calculating a second ratio of the levels measured in step c), and
[0111] e) comparing the second ratio to the first ratio.
[0112] In an embodiment, step e) of comparing the second ratio to
the first ratio is carried out by calculating a ratio of the second
ratio to the first ratio (or vice versa).
[0113] In a preferred embodiment of the methods of the present
invention, said methods further comprise the step of recommending
and/or initiating at least one suitable supportive measure, if it
is predicted that the subject is at risk of developing at least one
preeclampsia related adverse outcome after delivery of baby (or if
the subject is identified to be at risk of developing at least one
preeclampsia related adverse outcome after delivery of baby).
[0114] As discussed before, a subject suffering from at least one
preeclampsia related adverse outcome after delivery of baby
requires particular medical care. Thus, if a subject is identified
to be at risk of developing at least one preeclampsia related
adverse outcome after delivery of baby, in particular of developing
postpartum preeclampsia, postpartum eclampsia and/or postpartum
HELLP syndrome such an assessment can help to establish suitable
supportive measures for the subject in advance, i.e. before the
preeclampsia related adverse outcome after delivery of baby becomes
clinically apparent. Preferably, said at least one suitable
supportive measure is selected from the group consisting of: close
monitoring (in particular with respect to clinical symptoms of
postpartum HELLP syndrome, postpartum preeclampsia, or postpartum
eclampsia), admittance to an intensive care unit, administration of
corticosteroids, admission of magnesium sulfate, and administration
of blood pressure reducing agents and other specific measures
dependent on the adverse outcome of the mother Haram K, Svendsen E,
Abildgaard U. The HELLP syndrome: clinical issues and management. A
review. BMC Pregnancy and Childbirth 2009; 9(8).
http://dx.doi.org/10.1186/1471-2393-9-8 or DGGG. S1-Leitlinie:
Diagnostik und Therapie hypertensiver Schwangerschaftserkrankungen
der Deutschen Gesellschaft fu{umlaut over (r)} Gynakologie und
Geburtshilfe, see citation above.
[0115] Accordingly, the present invention further relates to a
method of initiating at least one suitable supportive measure in a
female subject after delivery of baby; said method comprising the
steps of the aforementioned methods of the present invention, the
further step of identifying a patient as being at risk of
developing at least one preeclampsia related adverse outcome after
delivery of baby, and the further step of initiating at least one
suitable supportive measure as outlined above.
[0116] If the subject is not at risk, the subject may be excluded
from said at least one supportive measure.
[0117] The present invention further relates to the (in vitro) use
of [0118] the biomarkers sFlt-1 (or Endoglin) and PlGF, or [0119]
an agent that (specifically) binds to sFlt-1 (or an agent that
(specifically) binds to Endoglin) and an agent that (specifically)
binds to PlGF in a first sample obtained from a female subject with
an uneventful pregnancy before delivery of baby and in a second
sample obtained from said female subject after delivery of baby for
predicting the risk of a female subject to develop at least one
preeclampsia related adverse outcome after delivery of baby.
[0120] The present invention further relates to the (in vitro) use
of [0121] the biomarkers sFlt-1 (or Endoglin) and PlGF, and/or
[0122] an agent that (specifically) binds to sFlt-1 (or an agent
that (specifically) binds to Endoglin) and an agent that
(specifically) binds to PlGF for the manufacture of a diagnostics
for predicting the risk of a female subject to develop at least one
preeclampsia related adverse outcome after delivery of baby, in
particular in a first sample obtained from a female subject with an
uneventful pregnancy before delivery of baby and in a second sample
obtained from said female subject after delivery of baby.
[0123] Preferably, the biomarkers or agents be used, as indicated
in the aforementioned method.
[0124] Preferably, a first and a second ratio of sFlt-1 or Endoglin
and PlGF (as described elsewhere herein) shall be calculated for
the first and the second sample and the ratios shall be compared,
in particular wherein an increase of the second ratio (or an
essentially unchanged ratio) as compared to the first ratio is
indicative for a subject who is at risk of developing at least one
preeclampsia related adverse outcome after delivery of baby, and/or
wherein a decrease of the second ratio as compared to the first
ratio is indicative for a subject who is not at risk of developing
a preeclampsia related adverse outcome after delivery of baby.
[0125] Preferably, the agent is a detection agent. In an
embodiment, the agent is an antibody such as a monoclonal or
polyclonal antibody.
[0126] Preferred diagnostic algorithms are disclosed herein
above.
[0127] Preferably, the agent is a detection agent. In an
embodiment, the agent is an antibody such as a monoclonal or
polyclonal antibody.
[0128] The present invention further relates to a device adapted
for predicting the risk of a female subject to develop at least one
preeclampsia related adverse outcome after delivery of baby, in
particular, by carrying out the aforementioned method, said device
comprising: [0129] a) an analyzer unit comprising an agent which
specifically binds to sFlt-1 and/or Endoglin and an agent which
specifically binds to PlGF, said unit being adapted for measuring
the level of sFlt-1 and/or Endoglin and the level of PlGF in a
first sample of a female subject obtained before delivery of baby
and a second sample of said female subject obtained after delivery
of baby; and [0130] b) an evaluation unit comprising a data
processor having implemented an algorithm for carrying out the
following steps of: [0131] i) calculating a first ratio from said
levels of sFlt-1 or Endoglin and PlGF determined in the first
sample and a second ratio from said levels of sFlt-1 or Endoglin
and PlGF determined in the second sample; and [0132] ii) comparing
the value of the said first and the said second ratio, and
optionally [0133] iii) predicting the risk of said subject to
develop at least one preeclampsia related adverse outcome after
delivery of baby, [0134] in particular whereby a subject is
predicted to be at risk for developing at least one preeclampsia
related adverse outcome after delivery of baby if the value of the
second ratio is increased (or essentially unchanged) as compared to
the value of the first ratio (and/or if the ratio of the second
ratio to the first ratio is equal to or larger than 1), and/or
whereby a subject is predicted to be not at risk for developing a
preeclampsia related adverse outcome after delivery of baby if the
value of the second ratio is decreased compared to the value of the
first ratio (and/or if the ratio of the second ratio to the first
ratio is lower than 1).
[0135] Optionally the algorithm for carrying out the following step
may further carry out the step of predicting the risk of developing
at least one preeclampsia related adverse outcome after delivery of
baby.
[0136] The term "device" as used herein relates to a system
comprising the aforementioned units operatively linked to each
other as to allow the diagnosis according to the methods of the
invention. Preferred agents (i.e. detection agents) which can be
used for the analyzer unit are disclosed elsewhere herein. The
analyzer unit, preferably, comprises said detection agents in
immobilized form on a solid support which is to be contacted to the
sample comprising the biomarkers the level of which is to be
determined. Moreover, the analyzer unit can also comprise a
detector which measures the level of detection agent which is
specifically bound to the biomarker(s). The measured level can be
transmitted to the evaluation unit. Said evaluation unit comprises
a data processing element, such as a computer, with an implemented
algorithm for carrying out a calculation of ratios (or of biomarker
levels), optionally a comparison of said calculated ratios and an
evaluation of the result of the comparison by implementation of an
computer based algorithm carrying out the steps of the method of
the present invention set forth elsewhere herein in detail. The
results may be given as output of parametric diagnostic raw data.
It is to be understood that these data will usually need
interpretation by the clinician. However, also envisaged are expert
system devices wherein the output comprises processed diagnostic
raw data the interpretation of which does not require a specialized
clinician.
[0137] According to some embodiments, an analyzer unit may be
configured for optical detection of an analyte, for example a
marker, with a sample. An exemplary analyzer unit configured for
optical detection comprises a device configured for converting
electro-magnetic energy into an electrical signal, which includes
both single and multi-element or array optical detectors. According
to the present disclosure, an optical detector is capable of
monitoring an optical electro-magnetic signal and providing an
electrical outlet signal or response signal relative to a baseline
signal indicative of the presence and/or concentration of an
analyte in a sample being located in an optical path. Such devices
may also include, for example, photodiodes, including avalanche
photodiodes, phototransistors, photoconductive detectors, linear
sensor arrays, CCD detectors, CMOS detectors, including CMOS array
detectors, photomultipliers, and photomultiplier arrays. According
to certain embodiments, an optical detector, such as a photodiode
or photomultiplier, may contain additional signal conditioning or
processing electronics. For example, an optical detector may
include at least one pre-amplifier, electronic filter, or
integrated circuit. Suitable pre-preamplifiers include, for
example, integrating, transimpedance, and current gain (current
mirror) pre-amplifiers.
[0138] Additionally, one or more analyzer unit according to the
instant disclosure may comprise a light source for emitting light.
For example, a light source of an analyzer unit may consist of at
least one light emitting element (such as a light emitting diode,
an electric powered radiation source such as an incandescent lamp,
an electroluminescent lamp, a gas discharge lamp, a high-intensity
discharge lamp, a laser) for measuring analyte concentrations with
a sample being tested or for enabling an energy transfer (for
example, through florescent resonance energy transfer or catalyzing
an enzyme).
[0139] Further, an analyzer unit of the system may include one or
more incubation units (for example, for maintaining a sample or a
reagent at a specified temperature or temperature range).
[0140] Additionally, an analyzer unit of the system disclosed
herein may comprise, or be operationally connected to, a reaction
vessel or cuvette feeding unit. Exemplary feeding units include
liquid processing units, such as a pipetting unit, to deliver
samples and/or reagents to the reaction vessels. The pipetting unit
may comprise a reusable washable needle, e.g. a steel needle, or
disposable pipette tips. The analyzer unit may further comprise one
or more mixing units, for example a shaker to shake a cuvette
comprising a liquid, or a mixing paddle to mix liquids in a
cuvette, or reagent container.
[0141] It follows from the above that according to some embodiments
of the instant disclosure, portions of some steps of methods
disclosed and described herein may be performed by a computing
device. A computing device may be a general purpose computer or a
portable computing device, for example. It should also be
understood that multiple computing devices may be used together,
such as over a network or other methods of transferring data, for
performing one or more steps of the methods disclosed herein.
Exemplary computing devices include desktop computers, laptop
computers, personal data assistants ("PDA"), such as BLACKBERRY
brand devices, cellular devices, tablet computers, servers, and the
like. In general, a computing device comprises a processor capable
of executing a plurality of instructions (such as a program of
software).
[0142] A computing device has access to a memory. A memory is a
computer readable medium and may comprise a single storage device
or multiple storage devices, located either locally with the
computing device or accessible to the computing device across a
network, for example. Computer-readable media may be any available
media that can be accessed by the computing device and includes
both volatile and non-volatile media. Further, computer
readable-media may be one or both of removable and non-removable
media. By way of example, and not limitation, computer-readable
media may comprise computer storage media. Exemplary computer
storage media includes, but is not limited to, RAM, ROM, EEPROM,
flash memory or any other memory technology, CD-ROM, Digital
Versatile Disk (DVD) or other optical disk storage, magnetic
cassettes, magnetic tape, magnetic disk storage or other magnetic
storage devices, or any other medium which can be used for storing
a plurality of instructions capable of being accessed by the
computing device and executed by the processor of the computing
device.
[0143] According to embodiments of the instant disclosure, software
may include instructions which, when executed by a processor of the
computing device, may perform one or more steps of the methods
disclosed herein. Some of the instructions may be adapted to
produce signals that control operation of other machines and thus
may operate through those control signals to transform materials
far removed from the computer itself. These descriptions and
representations are the means used by those skilled in the art of
data processing, for example, to most effectively convey the
substance of their work to others skilled in the art.
[0144] The plurality of instructions may also comprise an algorithm
which is generally conceived to be a self-consistent sequence of
steps leading to a desired result. These steps are those requiring
physical manipulations of physical quantities. Usually, though not
necessarily, these quantities take the form of electrical or
magnetic pulses or signals capable of being stored, transferred,
transformed, combined, compared, and otherwise manipulated. It
proves convenient at times, principally for reasons of common
usage, to refer to these signals as values, characters, display
data, numbers, or the like as a reference to the physical items or
manifestations in which such signals are embodied or expressed. It
should be borne in mind, however, that all of these and similar
terms are to be associated with the appropriate physical quantities
and are merely used here as convenient labels applied to these
quantities. According to some embodiments of the instant
disclosure, an algorithm for carrying out a comparison between a
determined level of one or more markers disclosed herein, and a
suitable reference, is embodied and performed by executing the
instructions. The results may be given as output of parametric
diagnostic raw data or as absolute or relative levels. According to
various embodiments of the system disclosed herein, a "diagnosis"
may be provided by the computing device of a system disclosed
herein based on said comparison of the calculated "level" to a
reference or a threshold. For example, a computing device of a
system may provide an indicator, in the form of a word, symbol, or
numerical value which is indicative of a particular diagnosis.
[0145] The computing device may also have access to an output
device. Exemplary output devices include fax machines, displays,
printers, and files, for example. According to some embodiments of
the present disclosure, a computing device may perform one or more
steps of a method disclosed herein, and thereafter provide an
output, via an output device, relating to a result, indication,
ratio or other factor of the method.
[0146] Furthermore, encompassed by the invention is a kit adapted
for carrying out the aforementioned method for predicting the risk
of a female subject for developing at least one preeclampsia
related adverse outcome after delivery of baby comprising i)
detection agents for determining the levels of the biomarkers
sFlt-1 and PlGF or ii) detection agents for determining the levels
of the biomarkers Endoglin and PlGF, or iii) detection agents for
determining the levels of the biomarkers sFlt-1, Endoglin and/or
PlGF as well as instructions for carrying out the said method.
[0147] The term "kit" as used herein refers to a collection of the
aforementioned components, preferably, provided in separately or
within a single container. The container also comprises
instructions for carrying out the method of the present invention.
These instructions may be in the form of a manual or may be
provided by a computer program code which is capable of carrying
out the comparisons referred to in the methods of the present
invention and to establish a diagnosis accordingly when implemented
on a computer or a data processing device. The computer program
code may be provided on a data storage medium or device such as a
optical storage medium (e.g., a Compact Disc) or directly on a
computer or data processing device. Further, the kit shall comprise
at least one standard for a reference as defined herein above, i.e.
a solution with a predefined level for the biomarker as referred to
herein representing a reference level.
[0148] In some embodiments, a kit disclosed herein includes at
least one component or a packaged combination of components for
practicing a disclosed method. By "packaged combination" it is
meant that the kits provide a single package that contains a
combination of one or more components, such as probes (for example,
an antibody), controls, buffers, reagents (for example, conjugate
and/or substrate) instructions, and the like, as disclosed herein.
A kit containing a single container is also included within the
definition of "packaged combination." In some embodiments, the kits
include at least one probe, for example an antibody (having
specific affinity for an epitope of a biomarker as disclosed
herein. For example, the kits may include an antibody that is
labelled with a fluorophore or an antibody that is a member of a
fusion protein. In the kit, the probe may be immobilized, and may
be immobilized in a specific conformation. For example, an
immobilized probe may be provided in a kit to specifically bind
target protein, to detect target protein in a sample, and/or to
remove target protein from a sample.
[0149] According to some embodiments, kits include at least one
probe, which may be immobilized, in at least one container. Kits
may also include multiple probes, optionally immobilized, in one or
more containers. For example, the multiple probes may be present in
a single container or in separate containers, for example, wherein
each container contains a single probe.
[0150] In some embodiments, a kit may include one or more
non-immobilized probe and one or more solid support that does or
does not include an immobilized probe. Some such embodiments may
comprise some or all of the reagents and supplies needed for
immobilizing one or more probes to the solid support, or some or
all of the reagents and supplies needed for binding of immobilized
probes to specific proteins within a sample.
[0151] In certain embodiments, a single probe (including multiple
copies of the same probe) may be immobilized on a single solid
support and provided in a single container. In other embodiments,
two or more probes, each specific for a different target protein or
a different form of a single target protein (such as a specific
epitope), a provided in a single container. In some such
embodiments, an immobilized probe may be provided in multiple
different containers (e.g., in single-use form), or multiple
immobilized probes may be provided in multiple different
containers. In further embodiments, the probes may be immobilized
on multiple different types of solid supports. Any combination of
immobilized probe(s) and container(s) is contemplated for the kits
disclosed herein, and any combination thereof may be selected to
achieve a suitable kit for a desired use.
[0152] A container of the kits may be any container that is
suitable for packaging and/or containing one or more components
disclosed herein, including for example probes (for example, an
antibody), controls, buffers, and reagents (for example, conjugate
and/or substrate). Suitable materials include, but are not limited
to, glass, plastic, cardboard or other paper product, wood, metal,
and any alloy thereof. In some embodiments, the container may
completely encase an immobilized probe(s) or may simply cover the
probe to minimize contamination by dust, oils, etc., and expose to
light. In some further embodiments, he kits may comprise a single
container or multiple containers, and where multiple containers are
present, each container may be the same as all other containers,
different than others, or different than some but not all other
containers.
[0153] The present invention also relates to a system for
predicting the risk of a female subject to develop at least one
preeclampsia related adverse outcome, comprising [0154] a) an
analyzer unit configured to contact, in vitro, a portion of a first
and second sample from the subject as set forth herein elsewhere
with i) an agent which specifically binds PlGF, and ii) an agent
which specifically binds sFlt-1, or an agent which specifically
binds Endoglin, [0155] b) an analyzer unit configured to detect a
signal from the portion of the sample from the subject contacted
with the agents, [0156] c) a computing device having a processor
and in operable communication with said analysis units, and [0157]
d) a non-transient machine readable media including a plurality of
instruction executable by a the processor, the instructions, when
executed calculate a first and a second ratio as set forth herein
elsewhere, and compare the first ratio with the second ratio,
thereby predicting the risk of a female subject to develop at least
one preeclamsia related adverse outcome.
[0158] All references referred to above are herewith incorporated
by reference with respect to their entire disclosure content as
well as their specific disclosure content explicitly referred to in
the above description.
EXAMPLES
[0159] The following Examples shall merely illustrate the
invention. They shall not be construed, whatsoever, to limit the
scope of the invention.
Example 1
Measurement of Serum Levels of PlGF, sFlt-1 and Endoglin
[0160] Serum levels of sFlt-1, PlGF and Endoglin were determined
using the commercially available immunoassays. In particular, the
following assays have been used.
[0161] sFlt-1 was determined with sandwich immunoassays using
analyzers from the Roche Elecsys.TM.- or cobas E.TM.-series. The
assay comprises two monoclonal antibodies specific for the
respective polypeptide. The first of these antibodies 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 the peptide to be determined 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 emitted amount of light
is dependent on the amount of sandwich complexes on the electrode.
The sFlt-1 test is commercially available from Roche Diagnostics
GmbH, Mannheim, Germany. Further details on the assay are found in
the package insert. The measuring range of sFlt-1 includes levels
between 10 to 85,000 pg/ml.
[0162] Endoglin was measured using the Quantikine.TM. Human
Endoglin/CD105 immunoassay which is commercially available from
R&D Systems, Inc, Minneapolis, US. This assay employs the
quantitative sandwich enzyme immunoassay technique. A monoclonal
antibody specific for Endoglin has been pre-coated onto a
microplate. Standards and samples are pipetted into the wells and
any Endoglin present is bound by the immobilized antibody. After
washing away any unbound substances, an enzyme-linked monoclonal
antibody specific for Endoglin is added to the wells. Following a
wash to remove any unbound antibody-enzyme reagent, a substrate
solution is added to the wells and color develops in proportion to
the level of Endoglin bound in the initial step. The color
development is stopped and the intensity of the color is measured.
Further details on the assay are found in the package insert. The
measuring range of Endoglin includes levels between 0.001 ng/L to
10 ng/ml.
[0163] PlGF was tested using two PlGF specific antibodies in a
sandwich immunoassay which is carried out on an Elecsys.TM.- or
cobas E.TM.-series analyzer (see above for details). The PlGF test
is commercially available from Roche Diagnostics GmbH, Mannheim,
Germany. Further details on the assay are found in the package
insert. The measuring range of PlGF includes levels of 3 to 10,000
pg/ml.
Example 2
[0164] Analysis of the biomarkers sFlt-1 and PlGF in outcome
patients which developed postpartum HELLP syndrome, postpartum
preeclampsia or postpartum eclampsia and in controls. R1 represents
the result of the ratio in the first sample; R2 corresponds to the
result of the ratio obtained from the second sample.
[0165] Women with postpartum HELLP syndrome/postpartum
preeclampsia/postpartum eclampsia [0166] 1) Woman with postpartum
HELLP syndrome: [0167] sFlt-1/PlGF ratio (R1)=44 [0168] sFlt-1/PlGF
ratio (R2)=64 [0169] R2/R1=65/44=1.45 (>=1) [0170] 2) Woman with
postpartum severe preeclampsia and associated hepatopathology:
[0171] sFlt-1/PlGF ratio (R1)=162 [0172] sFlt-1/PlGF ratio (R2)=283
[0173] R2/R1=283/162=1.74 (>=1)
Controls
[0174] Women with preeclampsia (clinical onset of disease before
delivery): [0175] 1) Woman with severe preeclampsia (onset of
disease and delivery in gestational week 33-36) [0176] sFlt-1/PlGF
ratio (R1)=101 [0177] sFlt-1/PlGF ratio (R2)=19 [0178]
R2/R1=19/101=0.18 (<1)
[0179] Women with no preeclampsia/eclampsia/HELLP syndrome: [0180]
1) Woman with elevated liver enzymes [0181] sFlt-1/PlGF ratio
(R1)=143 [0182] sFlt-1/PlGF ratio (R2)=73 [0183] R2/R1=73/143=0.51
(<1) [0184] 2) Another control woman [0185] sFlt-1/PlGF ratio
(R1)=132 [0186] sFlt-1/PlGF ratio (R2)=35 [0187] R2/R1=35/132=0.26
(<1)
[0188] In addition, the levels of sFlt-1 and PlGF in the sample
obtained after delivery were compared to the levels of sFlt-1 and
PlGF in the sample obtained before delivery. Interestingly, both
levels decreased after delivery in subjects with postpartum
preeclampsia related adverse outcomes. Based on the observed
decrease, it was not possible to establish a risk prediction for
the tested patients based on the levels of the single biomarker
sFlt-1 or PlGF alone respectively (as compared to the controls).
Thus, the ratio as disclosed herein is a reliable marker for
predicting the risk of postpartum preeclampsia related adverse
outcomes.
* * * * *
References