HK1228505B - Prediction of postpartum hellp syndrome, postpartum eclampsia or postpartum preeclampsia - Google Patents

Description

Prediction of postpartum HELLP syndrome, postpartum eclampsia, or postpartum preeclampsia

The present invention relates to methods for predicting the risk of a female subject who has had an uneventful pregnancy experiencing at least one adverse outcome associated with preeclampsia after delivery of an infant. The methods are based on determining the levels of i) sFlt-1 and PlGF or ii) endoglin and PlGF in a first sample obtained from the subject before delivery of the infant and a second sample obtained from the subject after delivery of the infant. Furthermore, the present invention encompasses devices and kits for practicing the methods of the invention.

Pregnancy can be complicated in various ways, which are associated with pregnancy-related mortality in pregnant women on the one hand, and increased morbidity and mortality in newborns on the other. Maternal mortality, at a rate of 14.5 per 100,000 live births, is more common in pregnant women over 39 years of age and can be caused by bleeding, thromboembolism, infection, cardiomyopathy, and cardiovascular and non-cardiovascular conditions, as well as hypertension, the most common of which is preeclampsia (Berg 2010, Obstetrics and Gynecology: 116: 1302-1309).

Preeclampsia affects approximately 2% to 8% of all pregnancies and is a major contributor to maternal and fetal mortality worldwide (Duley 2009, Semin Perinatol:33:130–37). Preeclampsia often develops during pregnancy. However, it can also occur postpartum, after the baby is born.

Preeclampsia is generally defined as pregnancy-related or induced hypertension. It is characterized by high blood pressure and proteinuria. Detailed information can also be found in standard medical textbooks and guidelines from various clinical societies, for example, Brown MA, Lindheimer MD, deSwiet M, Van Assche A, Moutquin JM: 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 für Gynäkologie und Geburtshilfe, AWMF online, AWMFRegister Nummer 015/018, Klasse S1.

In addition to preeclampsia, there are further preeclampsia-related adverse consequences that can occur after the baby is born, such as HELLP syndrome and eclampsia. All conditions are associated with adverse outcomes for the mother after birth.

HELLP syndrome is a life-threatening obstetric complication that involves hemolytic anemia, elevated liver function tests (LFTs), and a low platelet count. HELLP typically begins in the third trimester; however, up to 30% of all patients develop the syndrome postpartum, usually within 48 hours. The syndrome is unexpected, sudden, and fulminant in nature. In 20% of cases, there may be no evidence of preeclampsia before or during delivery, and all laboratory findings are 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).

Eclampsia is generally defined as a 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 usually occurs during or after the 20th week of gestation or in the postpartum period after the birth of the baby and delivery of the placenta.

There is a high unmet medical need for identifying women at risk for developing postpartum HELLP syndrome, eclampsia, or preeclampsia immediately after birth.

Placental growth factor (PlGF), soluble endoglin, and soluble fms-like tyrosine kinase 1 (sFlt-1) have been described as markers for diagnosing and predicting preeclampsia during pregnancy (see, e.g., WO2004/008946, WO2008/034750; Rana, 2007, Hypertension 50:137-142). The ratio of sFlt-1 to PlGF or endoglin to PlGF has been reported as a diagnostic or prognostic parameter for preeclampsia in pregnant women before delivery.

It is known in the literature that angiogenic and antiangiogenic factors decrease rapidly after delivery in healthy women as well as women with preeclampsia.

Wikström et al. examined the concentrations of sF1T-1 and PlGF before and after delivery in women with preeclampsia and controls and found a rapid decrease in both markers in all groups (Acta Obstericia et Gynecologia, 2008;87:146-153). However, women who presented with postpartum complications of preeclampsia were not included in this study.

Reddy et al. found that sFlt-1 (and activin A, but not soluble endoglin) levels increased during labor in women with preeclampsia compared with normal control women (PLoS ONE, 2009, 4(2), e4453). They found that sFlt-1 levels decreased within 24 hours in both groups. Women who presented with postpartum complications were not included in this study.

WO 2013/068475 describes a method for diagnosing pregnant women at risk of developing preeclampsia within a short timeframe (between about 15 and 34 weeks of pregnancy) by measuring the ratio (sFlt-1/PlGF) twice. If the ratio 2/ratio increases by at least about 3 times, the woman is at risk.

WO 2014/001244 describes a method for diagnosing whether a pregnant woman is at risk of developing pre-eclampsia (eclampsia and/or HELLP syndrome) within a short period of time (1-2 weeks); the pregnant subject is between about the 20th and about the 40th week of gestation.

Prager et al., 2013, monitored the sFlt-1/PlGF ratio in pregnant women who developed HELLP syndrome before delivery while on cortisone therapy. Women who developed postpartum HELLP syndrome were not included in this study (Prager, R; Eckart, A; Meint, P; Seelbach-Göbel, B: Verhalten der Angiogenesefaktoren (PlGF undsFlt-1) unter präpartaler Dexamethason-Therapie beim HELLP-Syndrom, Z Geburtshilfe Neonatol 2013; 217:Po01-6 DOI:10.1055/s-0033-1361384).

Early diagnosis of postpartum complications is important because the reported morbidity and mortality associated with these complications is high. For example, postpartum preeclampsia requires prompt treatment. If left untreated, postpartum preeclampsia can lead to epilepsy and other serious complications. Therefore, reliable assays for identifying subjects at risk for developing postpartum HELLP syndrome, postpartum eclampsia, and postpartum preeclampsia are not yet available, but remain highly desirable.

The technical problem underlying the present invention can be seen as providing means and methods for meeting the aforementioned need. This technical problem is solved by the embodiments characterized in the claims and below.

Advantageously, in the context of the studies underlying the present invention, it has been discovered that the sFlt-1/PlGF or endoglin/PlGF ratio in female subjects with an uneventful pregnancy serves as a biomarker for predicting the risk of said subject developing an adverse outcome associated with preeclampsia after the delivery of an infant, in particular developing postpartum preeclampsia, postpartum eclampsia, and/or postpartum HELLP syndrome. Significantly, an increase in the sFlt-1/PlGF or endoglin/PlGF ratio obtained after the delivery of an infant compared to a sample obtained before the delivery of the infant indicates a risk of developing an adverse outcome associated with preeclampsia after the delivery of the infant, whereas a decrease in the sFlt-1/PlGF or endoglin/PlGF ratio indicates that the subject is not at risk of developing an adverse outcome associated with preeclampsia after the delivery of the infant.

Thanks to the present invention, it is possible to more reliably assess the risk of at least one adverse outcome associated with preeclampsia after delivery of an infant based on reliable indicators. In addition, when the method of the present invention is applied, time-consuming, expensive, and cumbersome diagnostic measures can be avoided, and appropriate supportive measures can be initiated. Health care management will greatly benefit from the method of the present invention.

Thus, the present invention relates to a method for predicting the risk of a female subject developing at least one adverse outcome associated with preeclampsia after delivery of a baby (and thus suffering at least one adverse outcome associated with preeclampsia after delivery of a baby), said method comprising the steps of

a) Measured in a first sample obtained from a female subject with an uneventful pregnancy before delivery of the 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 measured in step a),

c) measuring the level of the biomarker as measured in step a) in a second sample obtained from said female subject after delivery of the baby,

d) calculating a second ratio of the levels measured in step c), and

e) comparing the second ratio to the first ratio.

In one embodiment, step e) of comparing the second ratio to the first ratio (or vice versa) is performed by calculating the ratio of the second ratio to the first ratio.

Preferably, the risk of the female subject experiencing at least one adverse outcome associated with preeclampsia after giving birth to the baby is predicted based on the results of the comparing step performed in step (e). Thus, the above method may further include the step of predicting (or providing a prediction of) the risk of the female subject experiencing at least one adverse outcome associated with preeclampsia after giving birth to the baby based on the results of the comparing step.

Preferably, the method of the present invention is an ex vivo or in vitro method. Moreover, in addition to those clearly mentioned above, it may also include other steps. For example, other steps may relate to sample pretreatment or evaluate the results obtained by the method. The method may be manually or assisted by automation. Preferably, the measuring step, the calculating step and the comparing step may be assisted in whole or in part by automation, for example, by a suitable robot and a sensing device for measuring, a computer-implemented calculation algorithm on a data processing device in the calculating step, or a comparison and/or diagnostic algorithm on a data processing device in the comparing step.

According to the present invention, it should be predicted that the female subject will develop, and therefore suffer from, the risk of at least one adverse consequence associated with preeclampsia after giving birth to the baby. The adverse consequences associated with preeclampsia that occur after giving birth to the baby are well known to technicians. As used herein, the term preferably refers to adverse consequences associated with preeclampsia that occur after pregnancy. Preferably, the adverse consequences associated with at least one preeclampsia after giving birth to the baby are selected from postpartum HELLP syndrome, postpartum preeclampsia and postpartum eclampsia, postpartum cerebral hemorrhage, postpartum renal failure, specifically postpartum acute renal failure, postpartum pulmonary edema, specifically acute postpartum pulmonary edema, postpartum cerebral edema, and postpartum liver rupture, disseminated intravascular coagulation (DIC) and postpartum maternal death. More preferably, the adverse consequences associated with at least one preeclampsia after giving birth to the baby are selected from postpartum HELLP syndrome, postpartum preeclampsia and postpartum eclampsia. Therefore, it is preferably predicted whether the female subject is at risk of developing postpartum HELLP syndrome, postpartum preeclampsia and/or postpartum eclampsia.

The term "at least one preeclampsia-related adverse outcome" refers to one preeclampsia-related adverse outcome or more than one, ie, two or three (or even more) preeclampsia-related outcomes (because, for example, eclampsia is often preceded by preeclampsia).

As used herein, the term "preeclampsia" refers to a medical condition characterized by hypertension and proteinuria. Before and after childbirth, that is, before and after the baby is born, preeclampsia may occur in a pregnant female subject. In the context of the present invention, it should be predicted that the subject will suffer from preeclampsia after childbirth, rather than the risk of preeclampsia during pregnancy. Most cases of postpartum preeclampsia occur within 48 hours after the baby is born. However, postpartum eclampsia sometimes occurs four to six weeks after the baby is born. This is referred to as late postpartum preeclampsia. Preferably, pregnancy-induced hypertension is identified as being present in a subject by two blood pressure measurements of 140 mmHg (systolic pressure) to 90 mmHg (diastolic pressure) or higher, wherein the two measurements are performed at least 6 hours apart. Proteinuria is preferably identified as being present in a 24 hour urine sample, specifically with 300 mg/dL or more protein. Also preferably, proteinuria is identified by protein dipstick analysis (if ≥2+), or if ≥30 mg/dL protein is present in a spot urine sample, or if the protein/creatinine ratio in the spot urine is ≥30 mg protein/mmol creatinine.

Preeclampsia can progress to eclampsia, a life-threatening condition characterized by the presence of tonic-clonic seizures or coma. Symptoms associated with severe preeclampsia are oliguria of less than 500 ml in 24 hours, cerebral or visual disturbances, pulmonary edema or cyanosis, pain in the upper abdomen or right upper quadrant, impaired liver function, and thrombocytopenia.

The term "HELLP syndrome" is well known in the art. HELLP syndrome is a life-threatening obstetric complication that is generally considered a complication of preeclampsia. Both conditions typically occur in the later stages of pregnancy or after delivery of the baby. In the context of the present invention, the risk of a female subject developing HELLP syndrome after delivery should be predicted. HELLP syndrome is associated with a high risk of adverse outcomes such as renal failure, subcapsular hematoma of the liver, recurrent preeclampsia, or even death. "HELLP" is an abbreviation for 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 between patients (patients often have no symptoms other than general abdominal pain), and early diagnosis is key to reducing morbidity. If left untreated, patients can become critically ill or die from liver rupture/hemorrhage or cerebral edema. In patients with possible HELLP syndrome, a battery of blood tests is performed: a complete blood count, a coagulation panel, liver enzymes, electrolytes, and renal function studies. Typically, the level of fibrin degradation products (FDPs) is measured, which can be elevated. Lactate dehydrogenase is a marker of hemolysis and is elevated (> 600 U/liter). Proteinuria is present but can be mild. Further details of preeclampsia and associated symptoms and subsequent diseases such as HELLP syndrome or eclampsia are found in standard medical textbooks or guidance from relevant medical societies. Details can be found, for example, 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 hypertensiverSchwangerschaftserkrankun-gen der Deutschen Gesellschaft für Gynäkologie undGeburtshilfe, see above citations.

As referred to herein, a "subject" is preferably a mammal. Mammals include, but are not limited to, domestic animals (e.g., cattle, 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 should be a female subject. The female subject should be pregnant when the first sample is obtained. However, the second sample should be obtained after the baby is born. The terms "subject" and "patient" are used interchangeably herein.

Preferably, the female subject should be a subject with an uneventful pregnancy. The term "undisturbed pregnancy" is well understood by the skilled person. In particular, it is envisaged that the subject with an uneventful pregnancy does not exhibit preeclampsia (in particular severe preeclampsia), eclampsia and/or HELLP syndrome during the course of pregnancy (i.e., during the present pregnancy). Therefore, the subject with an uneventful pregnancy preferably does not suffer from preeclampsia (in particular severe preeclampsia), eclampsia and/or HELLP syndrome before giving birth to the baby (in particular, during the present pregnancy). In particular, it is envisaged that the subject does not suffer from preeclampsia (in particular severe preeclampsia), eclampsia and HELLP syndrome before giving birth to the baby.

Therefore, when the first sample is obtained, the subject according to the present invention should preferably not have shown a clinical diagnosis of preeclampsia, eclampsia and/or HELLP syndrome before giving birth. However, the subject according to the present invention may show at least one symptom selected from upper abdominal pain, headache, visual disturbances, hypertension and edema, and may therefore be suspected of being at risk of developing (and therefore suffering from) at least one adverse consequence associated with preeclampsia after giving birth to the baby, in particular developing postpartum HELLP syndrome, postpartum preeclampsia and/or postpartum eclampsia. In one embodiment, the subject shows the at least one symptom shortly before giving birth to the baby, in particular the subject shows the at least one symptom when the first sample is obtained.

In addition, it is envisaged that the subject of an uneventful pregnancy suffers from mild preeclampsia before delivering the baby, i.e. during the present pregnancy. In this case, the risk refers to the occurrence of at least one severe preeclampsia-related adverse consequence after delivering the baby. Preferably, the severe preeclampsia-related adverse consequence 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 2 episodes of hypertension (specifically a blood pressure of ≥140/90 mm Hg) separated by at least 6 hours in a woman with normal blood pressure before 20 weeks of gestation, but without evidence of end-organ damage. 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, 2 times separated by at least 6 hours, more than 5 g of proteinuria in 24 hours or more than 3+ on 2 random urine samples collected at least 4 hours apart, oliguria (< 400 mL in 24 hours), persistent headache, upper abdominal pain and/or impaired liver function and thrombocytopenia. For the definition of this term, see, for example, Sibai et al. Lancet. 2005 Feb 26-Mar 4; 365(9461): 785-99, which is incorporated herein by reference for its entire disclosure.

Also preferably, the subject may be a person at risk of developing at least one adverse outcome associated with preeclampsia after the birth of a baby, in particular postpartum HELLP syndrome, postpartum preeclampsia and/or postpartum eclampsia. A person at risk is preferably a female subject older than 40 years of age and/or a female subject who is pregnant for the first time, has a family history of preeclampsia (e.g. preeclampsia in the mother or sister), has a previous history of preeclampsia in a previous pregnancy or after the birth of a previous baby, has a body mass index equal to or higher than 35 kg/m² at the time of first contact, has had multiple pregnancies or pre-existing vascular disease, such as hypertension or diabetes, for example as described in the NICE (National Institute for Health and Care Excellence) Antenatal Care guideline CG62, March 2008.

The delivery technique may be any technique deemed appropriate. Preferably, the delivery technique comprises one of an uninduced vaginal delivery, a cesarean section, and a medically induced delivery. In a preferred embodiment, a single infant is delivered. However, deliveries of more than one infant are also contemplated. Preferably, the infant is apparently healthy following delivery.

According to the method of the present invention, the risk of a female subject developing at least one adverse outcome associated with preeclampsia after giving birth to a baby, in particular postpartum HELLP syndrome, postpartum preeclampsia and/or postpartum eclampsia, and, therefore, the risk of said subject suffering from said adverse outcome should be predicted. Preferably, the prediction is made as to whether said adverse outcome occurs just after giving birth to a baby. The term "just after giving birth to a baby" with respect to said adverse outcome, in particular postpartum HELLP syndrome, postpartum preeclampsia and/or postpartum eclampsia is well understood by the skilled person. Preferably, the risk of developing at least one adverse outcome associated with preeclampsia is predicted within two weeks, more preferably within seven days, even more preferably within 72 hours or most preferably within 48 hours after giving birth to a baby. Preferably, said subject does not suffer from at least one adverse outcome associated with preeclampsia, in particular postpartum HELLP syndrome, postpartum preeclampsia and/or postpartum eclampsia, when the second sample is obtained.

As used herein, the term "prediction risk" preferably refers to the probability of evaluating whether at least one adverse consequence related to preeclampsia will occur in a subject after the delivery of the baby. More preferably, the risk/probability of adverse consequences related to at least one preeclampsia occurring (and therefore suffering) within a specific time window after the delivery of the baby is predicted. As described above, the prediction window is preferably a time interval of two weeks, seven days, 72 hours, 48 hours or any interrupted time range after the delivery of the baby. In a particularly preferred embodiment of the present invention, the prediction window is preferably a time interval of 48 hours. Preferably, the prediction window is calculated from the delivery of the baby. Also preferably, the prediction window is calculated from the time point at which the second sample has been obtained.

As will be understood by those skilled in the art, such predictions are generally not intended to be correct for 100% of subjects. However, the term requires that the prediction can be made in an appropriate and correct manner for a statistically significant portion of the subjects. Whether a portion is statistically significant can be determined without further fuss using various well-known statistical evaluation tools, such as determination of confidence intervals, determination of p-values, Student's t-tests, Mann-Whitney tests, and the like. For details, see 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-value is preferably 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the probabilities contemplated by the present invention allow predictions of increased, normal, or decreased risk to be corrected for at least 60%, at least 70%, at least 80%, or at least 90% of the subjects in a given cohort or population. The term preferably relates to predicting whether a subject is at increased risk or decreased risk immediately after delivery of a baby compared to the average risk in a population of female subjects of developing at least one adverse outcome associated with pre-eclampsia after delivery of a baby.

As used herein, the term "predicting the risk of developing at least one adverse consequence associated with preeclampsia after delivering a baby" means allocating the subject to be analyzed according to the method of the present invention into a group of subjects at risk of developing said at least one adverse consequence, or a group of subjects not at risk of developing at least one adverse consequence associated with preeclampsia. The risk of developing said at least one adverse consequence as mentioned in accordance with the present invention preferably means an increased risk of developing said at least one adverse consequence (within a prediction window). Preferably, the risk is increased compared to the average risk in a cohort of female subjects (i.e., a group of such subjects) just after delivering a baby. If the subject is not at risk of developing said adverse consequence associated with preeclampsia as mentioned in accordance with the present invention, then preferably, the risk of developing said adverse consequence should be reduced (within a prediction window). Preferably, the risk is reduced compared to the average risk in a cohort of female subjects just after delivering a baby. The subject at risk of developing said at least one adverse consequence preferably has a risk of developing said at least one adverse consequence of 80% or greater or more preferably 60% or greater, preferably just after delivering a baby. A subject not at risk of developing an adverse outcome associated with pre-eclampsia preferably has a risk of less than 20%, more preferably less than 10% or less or more preferably 5% or less of developing said at least one adverse outcome, preferably just after delivery of the baby.

According to the present invention, risk prediction can be provided. As used herein, the phrase "providing prediction" preferably refers to using the information or data generated about the first and second ratios in the patient sample to predict the risk of at least one adverse outcome related to preeclampsia occurring in the subject after giving birth to the baby. The information or data can be in any form, written, oral or electronic form. In some embodiments, the information or data generated are used to include communicating, presenting, reporting, storing, sending, transmitting, supplying, transmitting, distributing or a combination thereof. In some embodiments, communicating, presenting, reporting, storing, sending, transmitting, supplying, transmitting, distributing or a combination thereof is carried out by a computing device, an analyzer unit or a combination thereof. In some further embodiments, communicating, presenting, reporting, storing, sending, transmitting, supplying, transmitting, distributing or a combination thereof is carried out by a laboratory or a medical professional.

The term "sample" refers to a sample of a body fluid, a sample of a separated cell, or a sample from a tissue or organ. The sample of a body fluid can be obtained by well-known technology and includes samples of blood, plasma, serum, urine, lymph, sputum, ascites, or any other body secretions or derivatives thereof. Tissue or organ samples can be obtained from any tissue or organ by, for example, biopsy. Separated cells can be obtained from body fluids or tissues or organs by separation techniques such as centrifugation or cell sorting. For example, cells, tissues, or organ samples can be obtained from those cells, tissues, or organs that express or produce biomarkers. The sample can be frozen, fresh, fixed (e.g., formalin-fixed), centrifuged, and/or embedded (e.g., paraffin-embedded), etc. Before the level of the marker in the evaluation sample, the cell sample can of course be prepared and stored using various well-known collection techniques (e.g., nucleic acid and/or protein extraction, fixation, storage, freezing, ultrafiltration, concentration, evaporation, centrifugation, etc.). Similarly, biopsy samples can also be prepared and stored using techniques such as collection techniques, for example, fixation.

In one 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 a female subject. Also preferably, the sample is a urine sample.

According to the present invention, it is envisaged to measure the levels of the biomarkers as mentioned herein in the first and second samples from the female subject. The first sample should be obtained from the female subject before giving birth to the baby, in particular just before giving birth to the baby. Therefore, preferably, the first sample should be 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 giving birth to the baby. In addition, it is envisaged that the first sample is obtained within 12 hours before giving birth to the baby.

A "second sample" is preferably understood to be a sample obtained to reflect the change in the second ratio compared to the first ratio in the first sample. The second sample should be obtained after the first sample. Specifically, the second sample should be obtained after the delivery of the baby. Preferably, the second sample is obtained within 72 hours or 48 hours after the delivery of the baby, more preferably within 24 hours after the delivery of the baby, even more preferably within 16 hours, and most preferably within 12 hours after the delivery of the baby.

Preferably, the second sample is not obtained too soon after the first sample (to observe sufficiently significant changes to allow risk prediction). Thus, preferably the "second sample" is obtained no earlier than 10 hours, more preferably no earlier than 8 hours, or most preferably no earlier than 6 hours after the first sample. Thus, there should be a time interval of preferably at least 10 hours, more preferably at least 8 hours, and most preferably at least 6 hours between obtaining the first and second samples.

Also preferably, it is envisioned that the first sample is obtained no earlier than three hours before the delivery of the baby, and the second sample is obtained no earlier than three hours after the delivery of the baby. In addition, the first sample can be obtained no earlier than five hours before the delivery of the baby, and the second sample can be obtained no earlier than five hours after the delivery of the baby.

The term "delivery" is well understood by those skilled in the art in relation to the birth of a baby. This is the culmination of pregnancy, when one or more newborns are expelled from a woman's uterus. As used herein, the expression "delivery of a baby" preferably refers to the birth of a baby. More preferably, delivery of a baby is the point in time when the fetus is expelled from the subject's uterus. Most preferably, delivery of a baby is the point in time when the baby begins breathing. It is also contemplated that delivery of a baby is the point in time when the placenta is delivered.

In one embodiment of the invention, the baby is born without maternal or fetal complications.

It should be understood that the first and second samples are samples of the same type. For example, if the first sample is a serum sample, the second sample should also be a serum sample.

The term "measuring" the level of a marker as referred to herein refers to quantifying the biomarker, eg, determining the level of the biomarker in a sample, using an appropriate detection method as described elsewhere herein.

In one embodiment, the level of at least one biomarker is measured by contacting the sample with a detection reagent that specifically binds to the respective marker, thereby forming a complex between the reagent and the marker, detecting the level of the formed complex, and thereby measuring the level of the marker.

Biomarkers as mentioned herein can be detected using methods generally known in the art. The method of detection generally encompasses methods (quantitative methods) for determining the level of biomarkers in a quantitative sample. Technicians are generally known to be suitable for qualitative and/or quantitative detection of biomarkers in the following methods. Commercially available Westerns and immunoassays, such as ELISA, RIA, and fluorescence-based immunoassays can be conveniently used to measure, for example, protein in a sample. Further suitable methods for detecting biomarkers include measuring specific physical or chemical properties for a peptide or polypeptide, such as its precise molecular weight or NMR spectrum. The method includes, for example, a biosensor, an optical device coupled to an immunoassay, a biochip, an analytical device such as a mass spectrometer, an NMR analyzer, or a chromatography device. In addition, the method includes methods based on microplate ELISA, fully automated or robotic immunoassays (e.g., available on an Elecsys ^™ analyzer), CBA (enzymatic cobalt binding assay, such as available on a Roche-Hitachi ^™ analyzer), and latex agglutination assays (e.g., available on a Roche-Hitachi ^™ analyzer).

For the detection of biomarker proteins as mentioned herein, a wide range of immunoassay techniques using such assay formats are available, see, for example, U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653. These include single-site and two-site or "sandwich" assays of the non-competitive type, as well as traditional competitive binding assays. These assays also include direct binding of labeled antibodies to the target biomarker.

Sandwich assays are among the most useful and commonly used immunoassays.

Methods for measuring electrochemiluminescence are well known. Such methods exploit the ability of certain metal complexes to achieve an excited state by oxidation, from which the complexes decay to a ground state, emitting electrochemiluminescence. For a review, see Richter, M.M., Chem. Rev. 104 (2004) 3003-3036.

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 performance and ultra-high performance-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 fluorescence detection, and thin layer chromatography.

Preferably, measuring the level of a biomarker as mentioned herein comprises step (a) making the cell that can cause a cellular response contact with a peptide or polypeptide for a sufficient time period, the intensity of the cellular response indicating the level of the peptide or polypeptide, and (b) measuring the cellular response. For measuring cellular response, the sample or processed sample is preferably added to a cell culture, and internal or external cellular responses are measured. The cellular response can include the measurable expression of a reporter gene or the secretion of a material such as a peptide, polypeptide or micromolecule. Expression or material should generate an intensity signal associated with the level of the peptide or polypeptide.

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 a mass spectrum or NMR spectrum specific for the peptide or polypeptide.

Measuring the level of a peptide or polypeptide may preferably comprise the steps of (a) contacting the peptide with a specific binding agent, (b) (optionally) removing unbound binding agent, and (c) measuring the level of bound binding agent (i.e., the complex of binding agent formed in step (a)). According to a preferred embodiment, the steps of contacting, removing and measuring may be performed by an analyzer unit of the system disclosed herein. According to some embodiments, the steps may be performed by a single analyzer unit of the system or more than one analyzer unit that are in operable communication with each other. For example, according to a specific embodiment, the system disclosed herein may comprise a first analyzer unit for performing the contacting and removing steps and a second analyzer unit operably connected to the first analyzer unit by a transport unit (e.g., a robotic arm), which performs the measuring step.

The bound binding agent, i.e., the binding agent or binding agent/peptide complex, will generate an intensity signal. Binding according to the present invention includes covalent and non-covalent binding. The binding agent according to the present invention can be any compound, such as a peptide, polypeptide, nucleic acid, or small molecule that binds 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 containing binding domains for the peptide, and aptamers, such as nucleic acid or peptide aptamers. Methods for preparing such binding agents are well known in the art. For example, identification and production of suitable antibodies or aptamers are also provided by commercial suppliers. Those skilled in the art are familiar with methods for developing derivatives of such binding agents with higher affinity or specificity. For example, random mutations can be introduced into nucleic acids, peptides, or polypeptides. These derivatives can then be tested for binding according to screening procedures known in the art, such as phage display. Antibodies as mentioned herein include polyclonal and monoclonal antibodies, as well as fragments thereof, such as Fv, Fab, and F(ab) ₂ fragments that can bind to antigens or haptens. The present invention also includes single-chain antibodies and humanized hybrid antibodies in which the amino acid sequence of a non-human donor antibody showing the desired antigen specificity is combined with the sequence of a human acceptor antibody. The donor sequence will generally include at least the antigen-binding amino acid residues of the donor, but may also contain other structurally and/or functionally related amino acid residues of the donor antibody. Such hybrids can be prepared by several methods well known in the art. Preferably, the binding agent or reagent specifically binds to the peptide or polypeptide. Specific binding according to the present invention means that the ligand or reagent should not substantially bind to ("cross-react" with) another peptide, polypeptide or substance present in the sample to be analyzed. Preferably, the specifically bound peptide or polypeptide should bind with an affinity that is at least 3 times higher, more preferably at least 10 times higher and even more preferably at least 50 times higher than any other related peptide or polypeptide. Non-specific binding can be tolerated if it can still be clearly distinguished and measured, for example, based on its size on a protein 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, the method is semi-quantitative or quantitative. Other suitable techniques for determining polypeptides or peptides are described below.

The binding of the binding agent can be measured directly, for example, by NMR or surface plasmon resonance. According to a preferred embodiment, the measurement of the binding agent is realized by the analyzer unit of the system disclosed herein. Afterwards, the binding level measured can be calculated by the computing device of the system disclosed herein. If the binding agent also serves as a substrate for the enzymatic activity of the target peptide or polypeptide, the enzymatic reaction product can be measured (for example, the level of protease can be measured by, for example, measuring the level of the cleavage substrate on a Western blot). Alternatively, the binding agent can itself exhibit enzymatic properties, and the "binding agent/peptide or polypeptide" complex or the binding agent each bound by the peptide or polypeptide can be contacted with a suitable substrate, allowing detection by the generation of an intensity signal. For the measurement of the enzymatic reaction product, the level of the substrate is preferably saturated. The substrate can also be labeled with a detectable label before the reaction. Preferably, the sample is contacted with the substrate for a sufficient time period. A sufficient time period refers to the time required for the production of a product that can detect a preferably measurable level. Instead of measuring the level of the product, the time required for the occurrence of the product for a given (for example, detectable) level can be measured. Third, the binding agent can be covalently or non-covalently coupled to the label, allowing detection and measurement of the binding agent. Labeling can be accomplished by direct or indirect methods. Direct labeling involves direct (covalent or non-covalent) coupling of the label to the binding agent. Indirect labeling involves the binding (covalent or non-covalent) of a secondary binding agent to the first binding agent. The secondary binding agent should specifically bind to the first binding agent. The secondary binding agent can be coupled to a suitable label and/or be the target (receptor) of a tertiary binding agent that binds to the secondary binding agent. The use of secondary, tertiary or even higher order binding agents is generally used to increase the signal. Suitable secondary and higher order binding agents can include antibodies, secondary antibodies and the well-known streptavidin-biotin system (Vector Laboratories, Inc.). The binding agent or substrate can also be "labeled" with one or more labels as known in the art. Such a label can then be the target of a higher order binding agent. Suitable labels include biotin, digoxigenin, His-tags, glutathione-S-transferase, FLAG, GFP, myc-tags, influenza A virus hemagglutinin (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 the C-terminus. Suitable labels are any labels detectable by a suitable detection method. General labels include gold particles, latex beads, acridan esters, luminol, ruthenium, enzymatic activity labels, radioactive labels, magnetic labels ("e.g., magnetic beads" include paramagnetic and superparamagnetic labels), and fluorescent labels. Enzymatic activity labels include, for example, horseradish peroxidase, alkaline phosphatase, β-galactosidase, luciferase, and derivatives thereof. Suitable substrates for detection include diaminobenzidine (DAB), 3,3'-5,5'-tetramethylbenzidine, NBT-BCIP (4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate, available as a ready-to-use stock solution from Roche Diagnostics), CDP-Star™ (Amersham Bio-sciences), ECF™ (Amersham Biosciences). Suitable enzyme-substrate combinations can result in a colored reaction product, fluorescence or chemiluminescence, which can be measured according to methods known in the art (e.g., using photographic film or a suitable camera system). With regard to measuring enzymatic reactions, the criteria given above apply similarly. Common fluorescent labels include fluorescent proteins (e.g., GFP and its derivatives), Cy3, Cy5, Texas Red, fluorescein, and Alexa dyes (e.g., Alexa 568). Further fluorescent labels are available, for example, from Molecular Probes (Oregon). Quantum dots are also contemplated as fluorescent labels. The radioactive label can be detected by any known and suitable method, such as photographic film or phosphor imager.

The level of the peptide or polypeptide can also preferably be determined as follows: (a) a solid support comprising a binding agent for the peptide or polypeptide specified above is contacted with a sample comprising the peptide and polypeptide, and (b) the level of the peptide or polypeptide bound to the support is measured. The binding agent preferably selected from nucleic acids, peptides, polypeptides, antibodies and aptamers is preferably present on the solid support in an immobilized form. The materials used to make the solid support are well known in the art and include, in particular, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and/or silicon wafers and surfaces, nitrocellulose strips, membranes, sheets, duracytes, wells and walls of reaction trays, plastic tubing, etc. The binding agent or reagent can be bound to many different carriers. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified cellulose, polyacrylamide, agarose and magnetite. The nature of the carrier can be soluble or insoluble for the purposes of the present invention. Suitable methods for fixing/immobilizing the binding agent are well known and include, but are not limited to, ionic, hydrophobic, covalent interactions, etc. It is also contemplated to use "suspension arrays" as arrays according to the present invention (Nolan 2002, Trends Biotechnol. 20(1):9-12). In such suspension arrays, carriers such as microbeads or microspheres are present in suspension. The array consists of different microbeads or microspheres, which may be labeled and carry different binding agents. Methods for producing such arrays, for example based on solid phase chemistry and photolabile protective groups, are generally known (US 5,744,305).

In one embodiment of the present invention, the levels of biomarkers as mentioned herein are measured by using the assays described in the Examples section.

In another embodiment of the method of the present invention, the measurement in step a) (or in steps a) and c)) may be performed by an analyzer unit, in particular by an analyzer unit as defined elsewhere herein.

The term "binding agent" refers to a molecule comprising a binding moiety that specifically binds to a corresponding biomarker. Examples of "binding agents" are aptamers, antibodies, antibody fragments, peptides, peptide nucleic acids (PNAs), or compounds.

The term "specific binding" or "specifically binds" refers to a binding reaction in which a binding pair of molecules exhibits binding to each other under conditions in which they do not significantly bind to other molecules. When referring to a protein or peptide as a biomarker, the term "specific binding" or "specifically binds" refers to a binding reaction in which a binding agent binds to the corresponding biomarker with an affinity of at least ^10-7 M. The term "specific binding" or "specifically binds" preferably refers to an affinity of at least ^10-8 M or even more preferably at least ^10-9 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 a binding agent specific for the target molecule. Preferably, the level of binding to molecules other than the target molecule results in a binding affinity of only 10% or less, more preferably only 5% or less, as the affinity for the target molecule.

Examples of "binding agents" or "reagents" are nucleic acid probes, nucleic acid primers, DNA molecules, RNA molecules, aptamers, antibodies, antibody fragments, peptides, peptide nucleic acids (PNAs), or compounds. Preferred reagents are antibodies that specifically bind 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, as long as they exhibit the desired antigen-binding activity. Preferably, the antibody is a polyclonal antibody. More preferably, the antibody is a monoclonal antibody.

In one aspect, another binding agent that can be used can be an aptamer that specifically binds to at least one marker in a sample. When referring to a nucleic acid aptamer as a binding agent, the term "specific binding" or "specifically binds" refers to a binding reaction in which the nucleic acid aptamer binds to the corresponding target molecule with an affinity in the low nM to pM range.

In yet another aspect, before the level of the complex formed between the measuring bonding agent and at least one mark, the sample is removed from the formed complex. Therefore, in one aspect, the bonding agent can be immobilized on a solid support. In yet another aspect, the complex formed by the sample from the solid support can be removed by applying a washing solution. The level of the formed complex and the at least one mark present in the sample should be proportional. It should be understood that the specificity and/or sensitivity of the bonding agent to be applied define the ratio of at least one mark that can be specifically bound contained in the sample. A more detailed description of how to implement determination is also found elsewhere in this article. The level of the formed complex will be converted into the level of at least one mark, which reflects the level actually present in the sample. In one aspect, this type of level can be the level present in the sample basically, or in another aspect can be the level as its specific ratio caused by the relationship between the level present in the formed complex and the initial sample.

As used herein, the term "sFlt-1" refers to a polypeptide that is a soluble form of fms-like tyrosine kinase 1. This polypeptide is also known in the art as soluble VEGF receptor 1 (sVEGF R1) (see, e.g., Sunderji 2010, Am J Obstet Gynecol 202:40e1-7). It was identified in conditioned medium from 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 considerable affinity. Human sFlt-1 has been shown to form a stable complex with the extracellular domain of KDR/Flk-1 in vitro. Preferably, sFlt-1 refers to human sFlt-1 as described in Kendall 1996, Biochem Biophs Res Commun 226(2):324-328; for amino acid sequences, see also, for example, Genebank accession numbers P17948, GI:125361 (human sFlt-1) and BAA24499.1, GI:2809071 (mouse sFlt-1) (Genbank is available from NCBI, USA at www.ncbi.nlm.nih.gov/entrez). The term also encompasses variants of the aforementioned human sFlt-1 polypeptide. Such variants have at least the same essential biological and immunological properties as the aforementioned sFlt-1 polypeptide. Specifically, they have the same essential biological and immunological properties if they are detectable by the same specific assays mentioned herein, such as by an ELISA assay using polyclonal or monoclonal antibodies that specifically recognize the sFlt-1 polypeptide. Furthermore, it is understood that variants according to the present invention may have an amino acid sequence that differs by 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 to the entire length of a specific sFlt-1 polypeptide, preferably human sFlt-1. 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 determined by comparing two optimally aligned sequences over a comparison window, wherein segments of the amino acid sequence in the comparison window may contain additions or deletions (e.g., gaps or overhangs) for optimal alignment compared to a reference sequence (which does not contain additions or deletions). The percentage is calculated by determining the number of positions where the identical amino acid residue is present in the two sequences to generate the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to generate the percentage of sequence identity. Can be by Smith1981, Add.APL.Math.2:482 disclosed local homology algorithm, by Needleman 1970, J.Mol.Biol.48:443 homology comparison algorithm, by Pearson 1988, the method for the retrieval similarity of Proc.Natl.Acad Sci.(USA)85:2444, by the computerized realization (Wisconsin GeneticsSoftware Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, GAP, BESTFIT, BLAST, FAST, PASTA and TFASTA) of these algorithms or by visual inspection carry out the best alignment for the sequence compared.In view of having identified two sequences for comparison, preferably utilize GAP and BESTFIT to determine their best alignment, therefore determine the identity degree.Preferably, use the default value of gap weight 5.00 and gap length weight 0.30. The variants mentioned above may be allelic variants or any other species-specific homologs, paralogs, or orthologs. The variants mentioned above may be allelic variants or any other species-specific homologs, paralogs, or orthologs. Furthermore, the variants mentioned herein include fragments or subunits of a particular sFlt-1 polypeptide or variants of the aforementioned types, as long as these fragments possess the basic immunological and biological properties mentioned above. Such fragments may be, for example, degradation products of the sFlt-1 polypeptide. Variants are considered to possess the same basic biological and immunological properties if they can be detected by the same specific assays mentioned in this specification, such as an ELISA assay using polyclonal or monoclonal antibodies that specifically recognize the sFlt-1 polypeptide. Preferred assays are described in the accompanying Examples. Further included are variants that differ due to post-translational modifications such as phosphorylation or myristylation. sFlt-1 can be detected in a bound or free form or as the total sFlt-1 level in a sample.

As used herein, the term "endoglin" refers to a polypeptide having a molecular weight of 180 kDa in the non-reduced state, 95 kDa after reduction, and 66 kDa in its reduced and N-deglycosylated form. Preferably, the term "endoglin" refers to soluble endoglin. This polypeptide is capable of forming dimers and binds to TGF-β and the TGF-β receptor. Preferably, endoglin refers to human endoglin. More preferably, human endoglin has the amino acid sequence shown in Genebank Accession No. AAC63386.1, GI:3201489. Two endoglin isoforms have been described, S-endoglin and L-endoglin. L-endoglin consists of a 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. The soluble endoglin as referred to herein is preferably described in EP 1 804 836 B1. Furthermore, it is understood that variants according to the present invention may have an amino acid sequence that 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 to the amino acid sequence of the specific endoglin. Variants may be allelic variants, splice variants or any other species-specific homologs, paralogs or orthologs. Furthermore, variants referred to herein include fragments of the specific endoglin or variants of the aforementioned types, as long as these fragments have the basic immunological and biological properties as mentioned above. Such fragments may be, for example, degradation products of endoglin. Variants are considered to share the same basic biological and immunological properties if they are detectable by the same specific assays mentioned in this specification, such as by an ELISA assay using polyclonal or monoclonal antibodies that specifically recognize the endoglin polypeptide. Preferred assays are described in the accompanying Examples. Further included are variants that differ due to post-translational modifications such as phosphorylation or myristylation. Endoglin can be detected in bound or free form or as total endoglin levels in a sample.

As used herein, the term "P1GF (placental growth factor)" preferably refers to a placenta-derived growth factor, which is a polypeptide having a length of 149 amino acids and a high degree of homology to the platelet-derived growth factor-like region of human vascular endothelial growth factor (VEGF). Like VEGF, P1GF has angiogenic activity in vitro and in vivo. For example, biochemical and functional characterization of P1GF derived from transfected COS-1 cells revealed that it is a glycosylated dimer secreted protein capable of stimulating endothelial cell growth in vitro (Maqlione 1993, Oncogene 8(4):925-31). Preferably, P1GF refers to human P1GF, more preferably, to human P1GF having an amino acid sequence as shown in Genebank accession number P49763, GI:17380553. The term encompasses variants of the specific human P1GF. Such variants have at least the same basic biological and immunological properties as the specific P1GF polypeptide. If variants can be detected by the same specific assays mentioned in this specification, such as by ELISA assays using polyclonal or monoclonal antibodies that specifically recognize the PIGF polypeptide, they are considered to have the same basic biological and immunological properties. Preferred assays are described in the accompanying examples. In addition, it should be understood that variants as mentioned in accordance with the present invention should have an amino acid sequence that 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 to the amino acid sequence of the specific PIGF polypeptide. The degree of identity between two amino acid sequences can be determined by algorithms well known in the art and described elsewhere herein. The variants mentioned above may be allelic variants or any other species-specific homologs, paralogs or orthologs. Moreover, the variants mentioned herein include fragments of the specific PIGF polypeptide or variants of the aforementioned types, as long as these fragments have the basic immunological and biological properties mentioned above. Such fragments can be, for example, degradation products or splice variants of the PIGF polypeptide. Further included are variants that differ due to post-translational modifications such as phosphorylation or myristylation. PIGF can be detected in a bound or free form or as the total PIGF level in a sample.

As used herein, the term "level" encompasses the absolute amount of a biomarker as mentioned herein, the relative amount or concentration of the biomarker, and any value or parameter related thereto or that can be derived therefrom. Such values or parameters include intensity signal values from all specific physical or chemical properties, such as intensity values in a mass spectrum or NMR spectrum, obtained from the peptide by direct measurement. Furthermore, all values or parameters obtained by indirect measurements indicated elsewhere in this specification are encompassed, such as the amount of response to the peptide measured from a biological readout system or the intensity signal obtained from a specifically bound ligand. It should be understood that values related to the aforementioned amounts or parameters can also be obtained by all standard mathematical operations.

As used herein, the term "comparison" refers to comparing the ratio (first ratio) of the level of a biomarker as mentioned herein in the first sample from a subject with the ratio (second ratio) of the level of the biomarker in the second sample from a subject. It should be understood that comparison as used herein generally refers to comparing corresponding parameters or values, for example, comparing an absolute amount with an absolute reference amount, while comparing a concentration with a reference concentration, or comparing the intensity signal of the biomarker obtained from a sample with the intensity signal of the same type obtained from a reference sample. The comparison can be implemented manually or computer-assisted. Therefore, the comparison can be implemented by a computing device (for example, a computing device of a system disclosed herein). The value of the (first) ratio in the first sample from a subject and the value of the (second) ratio in the second sample can, for example, be compared with each other, and the comparison can be automatically implemented by a computer program executing an algorithm for comparison. The computer program implementing the assessment will provide an expected evaluation of a suitable output format. For computer-assisted comparison, the value of the determination ratio in the second sample can be compared with the value of the ratio in the first sample stored in a database by a computer program. The computer program can further evaluate the result of the comparison, i.e., automatically provide an expected evaluation of a suitable output format. For computer-assisted comparison, the value of the determined ratio in the second sample can be compared with the value of the ratio in the first sample stored in a database by a computer program. The computer program can further evaluate the results of the comparison, i.e. automatically provide the desired evaluation in a suitable output format.

As referred to herein, the term "calculating a first ratio" or "calculating a second ratio" refers to calculating the ratio of the level of sFlt-1 or endoglin to the level of PlGF by dividing by the levels or by performing any other similar mathematical calculation that places the level of sFlt-1 or endoglin in relation to 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, calculating the ratio of the level of sFlt-1 or endoglin to the level of PlGF). Also preferably, the level of PlGF is divided by the level of sFlt-1 or endoglin in order to calculate the ratio (thus, calculating the ratio of the level of PlGF to the level of sFlt-1 or endoglin). The calculations are performed for the respective levels determined separately in the first and second samples to obtain the first and second ratios, respectively. The calculations may be performed simultaneously or at different times.

If the method comprises a comparison of the second ratio with the first ratio, preferably the following applies:

In one embodiment, the first and second ratios are the ratios of sFlt-1 to PlGF, or the ratios of endoglin to PlGF. Preferably, an increase in the second ratio (or the substantially unchanged second ratio) compared to the first ratio indicates a subject at risk of developing at least one adverse outcome associated with preeclampsia after delivering the baby, and/or a decrease in the second ratio compared to the first ratio indicates a subject not at risk of developing adverse outcomes associated with preeclampsia after delivering the baby. Also preferably, if the second ratio increases compared to the first ratio, or if the second ratio is substantially the same as the first ratio, the subject is at risk of developing at least one adverse outcome associated with preeclampsia after delivering the baby, and if the second ratio decreases compared to the first ratio, the subject is not at risk of developing adverse outcomes associated with preeclampsia after delivering the baby.

In another embodiment, the first and second ratios are the ratios of PlGF to sFlt-1, or the ratios of PlGF to endoglin. Preferably, a decrease in the second ratio (or the substantially unchanged second ratio) compared to the first ratio indicates a subject at risk of developing at least one adverse outcome associated with preeclampsia after delivering the baby, and/or an increase in the second ratio compared to the first ratio indicates a subject not at risk of developing adverse outcomes associated with preeclampsia after delivering the baby. Also preferably, if the second ratio decreases compared to the first ratio, or if the second ratio is substantially the same as the first ratio, the subject is at risk of developing at least one adverse outcome associated with preeclampsia after delivering the baby, and if the second ratio increases compared to the first ratio, the subject is not at risk of developing adverse outcomes associated with preeclampsia after delivering the baby.

The term "substantially unchanged" is well known in the art and is understood by those skilled in the art of diagnosis. The term refers to a small change in the second ratio compared to the first ratio, such as a change of less than 3 or 7%. In one embodiment, the term refers to a ratio that is unchanged.

If step e) of comparing the second ratio to the first ratio is performed by calculating the ratio of the second ratio to the first ratio) (or vice versa), preferably the following applies:

If the first and second ratios are the ratios of sFlt-1 to PlGF, or the ratios of endoglin to PlGF, the following applies: Preferably, a ratio equal to or greater than 1 indicates a subject who is at risk of developing at least one adverse outcome associated with preeclampsia after delivery of the infant, and a ratio below 1 indicates a subject who is not at risk of developing an adverse outcome associated with preeclampsia after delivery of the infant.

If the first and second ratios are the ratios of PlGF to sFlt-1, or the ratios of PlGF to endoglin, the following applies: preferably, a ratio equal to or less than 1 indicates a subject who is at risk of developing at least one adverse outcome associated with preeclampsia after delivery of the infant, and a ratio greater than 1 indicates a subject who is not at risk of developing an adverse outcome associated with preeclampsia after delivery of the infant.

According to the present invention, the terms "increase" and "decrease" preferably refer to a statistically significant increase and decrease, respectively. Specifically, a statistically significant increase (or decrease) is an increase (or decrease) of a magnitude that is considered statistically significant for risk prediction. The terms "significant" and "statistically significant" are known to those skilled in the art. Those skilled in the art can readily determine whether an increase or decrease is statistically significant using various well-known statistical evaluation tools, including those mentioned herein.

It has been discovered in the course of the present invention that a preferred increase in 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, indicating that the subject is at risk of developing at least one adverse outcome associated with preeclampsia after delivering the baby, is preferably an increase of at least 3%, more preferably at least 10%, and even more preferably at least 20%, and most preferably at least 30%.

It has been discovered in the course of the present invention that a preferred reduction in 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, indicating that a subject is not at risk of developing an adverse outcome associated with preeclampsia after delivering the baby, is preferably a reduction of at least 10%, more preferably at least 20%, and even more preferably at least 30%, and most preferably at least 40%.

It is to be understood that the definitions and interpretations of the terms given above and below apply accordingly to all embodiments described in the present specification and the appended claims.

The present invention further relates to a method for distinguishing a subject at risk of developing at least one adverse outcome associated with preeclampsia after delivering a baby from a subject not at risk of developing at least one adverse outcome associated with preeclampsia after delivering a baby, the method comprising the steps of

a) Measured in a first sample obtained from a female subject with an uneventful pregnancy before delivery of the 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 measured in step a),

c) measuring the level of the biomarker as measured in step a) in a second sample obtained from said female subject after delivery of the baby,

d) calculating a second ratio of the levels measured in step c), and

e) comparing the second ratio to the first ratio.

In one embodiment, step e) of comparing the second ratio to the first ratio (or vice versa) is performed by calculating the ratio of the second ratio to the first ratio.

The present invention further relates to a method for identifying a subject at risk of experiencing at least one adverse outcome associated with preeclampsia following delivery of a baby, the method comprising the steps of

a) Measured in a first sample obtained from a female subject with an uneventful pregnancy before delivery of the 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 measured in step a),

c) measuring the level of the biomarker as measured in step a) in a second sample obtained from said female subject after delivery of the baby,

d) calculating a second ratio of the levels measured in step c), and

e) comparing the second ratio to the first ratio.

In one embodiment, step e) of comparing the second ratio to the first ratio (or vice versa) is performed by calculating the ratio of the second ratio to the first ratio.

In a preferred embodiment of the method of the present invention, the method further comprises the step of recommending and/or initiating at least one appropriate supportive measure if the subject is predicted to be at risk of developing at least one adverse outcome associated with preeclampsia after delivery of the baby (or if the subject is identified as being at risk of developing at least one adverse outcome associated with preeclampsia after delivery of the baby).

As previously discussed, subjects who suffer at least one adverse outcome associated with preeclampsia after delivery of a baby require special medical care. Therefore, if a subject is identified as being at risk for developing at least one adverse outcome associated with preeclampsia after delivery of a baby, particularly developing postpartum preeclampsia, postpartum eclampsia, and/or postpartum HELLP syndrome, such an evaluation can help establish appropriate supportive measures for the subject in advance, i.e., before the adverse outcome associated with preeclampsia after delivery of the baby becomes clinically apparent. Preferably, the at least one appropriate supportive measure is selected from the group consisting of: close monitoring (particularly with respect to clinical symptoms of postpartum HELLP syndrome, postpartum preeclampsia, or postpartum eclampsia), admission to an intensive care unit, administration of corticosteroids, incorporation of magnesium sulfate, and administration of antihypertensive agents and other specific measures depending on the adverse outcome for 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 Therapiehypertensiver Schwangerschaftserkrankungen der Deutschen Gesellschaft für Gynäkologie und Geburtshilfe, see citation above.

Thus, the present invention further relates to a method of initiating at least one appropriate supportive measure in a female subject after delivery of a baby; said method comprising the steps of the above-described method of the present invention, the further step of identifying the patient as being at risk of developing at least one adverse outcome associated with preeclampsia after delivery of the baby, and the further step of initiating at least one appropriate supportive measure as outlined above.

If the subject is not at risk, the subject may be excluded from the at least one supportive measure.

The present invention further relates to

• Biomarkers sFlt-1 (or endoglin) and PlGF, or

• An agent that (specifically) binds sFlt-1 (or an agent that (specifically) binds endoglin) and an agent that (specifically) binds PlGF

(In vitro) use of a first sample obtained from a female subject with an uneventful pregnancy before delivery of a baby and a second sample obtained from said female subject after delivery of a baby for predicting the risk of the female subject experiencing at least one adverse outcome associated with preeclampsia after delivery of a baby.

The present invention further relates to

• Biomarkers sFlt-1 (or endoglin) and PlGF, and/or

• An agent that (specifically) binds sFlt-1 (or an agent that (specifically) binds endoglin) and an agent that (specifically) binds PlGF

Specifically, a first sample obtained from a female subject with an uneventful pregnancy before giving birth to a baby and a second sample obtained from the female subject after giving birth to a baby are used to prepare an (in vitro) use of a diagnostic agent for predicting the risk of a female subject developing at least one adverse outcome associated with preeclampsia after giving birth to a baby.

Preferably, biomarkers or reagents as indicated in the above methods are used.

Preferably, a first and a second ratio of sFlt-1 or endoglin and PlGF (as described elsewhere herein) should be calculated for the first and second samples, and the ratios should be compared, specifically, wherein an increase in the second ratio (or a substantially unchanged ratio) compared to the first ratio indicates a subject at risk of developing at least one adverse outcome associated with preeclampsia after delivery of the infant, and/or wherein a decrease in the second ratio compared to the first ratio indicates a subject not at risk of developing an adverse outcome associated with preeclampsia after delivery of the infant.

Preferably, the reagent is a detection reagent. In one embodiment, the reagent is an antibody, such as a monoclonal antibody or a polyclonal antibody.

Preferred diagnostic algorithms are disclosed herein above.

Preferably, the reagent is a detection reagent. In one embodiment, the reagent is an antibody, such as a monoclonal antibody or a polyclonal antibody.

The present invention further relates to a device adapted to predict the risk of at least one adverse outcome associated with preeclampsia in a female subject after delivery of a baby, in particular by implementing the above method, the device comprising:

a) an analyzer unit comprising a reagent that specifically binds sFlt-1 and/or endoglin and a reagent that specifically binds PlGF, said unit being adapted to measure the level of sFlt-1 and/or endoglin and the level of PlGF in a first sample obtained from a female subject before delivery of a baby and in a second sample obtained from said female subject after delivery of the baby; and

b) an evaluation unit comprising a data processor in which an algorithm for carrying out the following steps has been implemented:

i) calculating a first ratio from said levels of sFlt-1 or endoglin and PlGF determined in a first sample, and calculating a second ratio from said levels of sFlt-1 or endoglin and PlGF determined in a second sample; and

ii) comparing the values of said first and said second ratios, and optionally

iii) predicting the risk of said subject experiencing at least one adverse outcome associated with preeclampsia following delivery of an infant,

Specifically, if the value of the second ratio increases (or remains essentially unchanged) 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 greater than 1), it is predicted that the subject is at risk of experiencing at least one adverse outcome related to preeclampsia after delivering the baby, and/or if the value of the second ratio decreases compared to the value of the first ratio (and/or if the ratio of the second ratio to the first ratio is less than 1), it is predicted that the subject is not at risk of experiencing an adverse outcome related to preeclampsia after delivering the baby.

Optionally, the algorithm for performing the following steps may further perform the step of predicting the risk of experiencing at least one pre-eclampsia-related adverse outcome following delivery of the infant.

As used herein, term "device" refers to a system comprising the aforementioned units that are operably connected to each other to allow the diagnosis according to the method of the present invention. Preferred reagents (i.e., detection reagents) that can be used for analyzer units are disclosed elsewhere herein. The analyzer unit preferably comprises the detection reagent in an immobilized form on a solid support to be contacted with a sample, the sample and the biomarker comprising its level to be determined. Moreover, the analyzer unit may also comprise a detector for measuring the level of the detection reagent for specific binding biomarkers. The level of measurement may be transferred to an evaluation unit. The evaluation unit comprises a data processing element such as a computer, which has an implementation algorithm, the implementation algorithm being used to implement the calculation of a ratio (or the ratio of biomarker levels), optionally comparing the calculated ratio and evaluating the result of the comparison by implementing a computer-based algorithm for the method steps of the present invention described in detail elsewhere herein. The result can be given as the output of parameter diagnostic raw data. It should be understood that these data will generally require the explanation of a clinician. However, it is also envisioned that an expert system device is provided, wherein the output comprises processed diagnostic raw data, and its explanation does not require a specialized clinician.

According to some embodiments, the analyzer unit can be configured to detect analytes in optical samples, such as markers. The exemplary analyzer unit configured for optical detection includes a device configured to convert electromagnetic energy into electrical signals, including single elements and multi-element or array optical detectors. According to the present disclosure, the optical detector can monitor light electromagnetic signals and provide an electrical outlet signal (electrical outlet signal) or a response signal relative to a baseline signal indicating the presence and/or concentration of analytes in the sample located in the light path. Such devices can also include, for example, photodiodes, including avalanche photodiodes (avalanche photodiodes), phototransistors, photoconductivity detectors, linear sensor arrays, CCD detectors, CMOS detectors, including CMOS array detectors, photomultiplier tubes, and photomultiplier tube arrays. According to certain embodiments, optical detectors, such as photodiodes or photomultiplier tubes, can contain additional signal conditioning or processing electrical components. For example, the optical detector can include at least one preamplifier, electronic filter, or integrated circuit. Suitable preamplifiers include, for example, integrated, transimpedance, and current gain (current mirror) preamplifiers.

In addition, one or more analyzer units according to the present disclosure may include a light source for emitting light. For example, the light source of the analyzer unit may be composed of at least one light emitting element (such as a light emitting diode, an electric power emitting source such as an incandescent lamp, an electroluminescent lamp, a gas discharge lamp, a high intensity discharge lamp, a laser) for measuring the concentration of an analyte in a sample to be tested, or enabling energy conversion (e.g., by fluorescence resonance energy transfer or catalytic enzymes).

Furthermore, the analyzer unit of the system may include one or more incubation units (eg, for maintaining samples or reagents at a specific temperature or temperature range).

In addition, the analyzer unit of the system disclosed herein may include or be operably connected to a reaction vessel or a cuvette delivery unit. Exemplary delivery units include liquid processing units, such as pipetting units, for delivering samples and/or reagents to the reaction vessel. The pipetting unit may include a reusable washable needle, such as a steel needle, or a disposable pipetting head. The analyzer unit may also include one or more mixing units, such as an oscillator for oscillating a cuvette containing a liquid, or a stirring paddle for mixing the liquid in the cuvette or reagent container.

According to the above description, according to some embodiments of the present disclosure, parts of some steps of the methods disclosed and described herein can be performed by a computing device. The computing device can be, for example, a general-purpose computer or a portable computing device. It should also be understood that multiple computing devices can be used together, such as via a network or other methods of transferring data, for implementing one or more steps of the methods disclosed herein. Exemplary computing devices include desktop computers, portable computers, personal data assistants ("PDAs"), such as BLACKBERRY brand devices, mobile phone devices (cellular devices), tablet computers, servers, etc. Typically, a computing device includes a processor capable of executing multiple instructions (such as software programs).

The computing device is able to access the memory. The memory is a computer-readable medium and can include a single storage device or multiple storage devices, for example, which are locally located on the computing device or can access the computing device across a network. The computer-readable medium can be any accessible medium that can be accessed by the computing device, and includes both stable and unstable media. In addition, the computer-readable medium can be one or both of removable and non-removable media. By way of example and not limitation, the computer-readable medium can include computer storage media. Exemplary computer storage media include, but are not limited to, RAM, ROM, EEPROM, flash memory or any other storage technology, CD-ROM, digital versatile disc (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, disk storage or other magnetic storage devices, or any other medium that can be used to store multiple instructions that can be accessed by the computing device and executed by the processor of the computing device.

According to embodiments of the present disclosure, software may include instructions that, when executed by a processor of a computing device, may perform one or more steps of the methods disclosed herein. Some instructions may be adapted to generate signals that control the operation of other machines, and thus may operate through those control signals to transform materials that are remote from the computer itself. These descriptions and representations are 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.

The multiple instructions may also include such an algorithm, which is generally understood to be a self-consistent sequence of steps that leads to a desired result. These steps are those that require physical manipulation of physical quantities. Typically, although not necessarily, these quantities take the form of electrical or magnetic pulses or signals that can be stored, transferred, converted, combined, compared, and otherwise manipulated. Sometimes, primarily for common usage reasons, it is convenient to refer to these signals as values, characters, displayed data, numbers, etc. as references to the physical items or manifestations in which these signals are embodied or represented. However, it should be remembered that all of these and similar terms should be related to appropriate physical quantities and are used herein only as convenient labels for applying to these quantities. According to some embodiments of the present disclosure, an algorithm for comparing the measured levels of one or more markers disclosed herein with a suitable reference is embodied and performed by executing instructions. The results can be output as parameter diagnostic raw data or given as absolute or relative levels. According to multiple embodiments of the system disclosed herein, a "diagnosis" can be provided by a computing device of the system disclosed herein based on a comparison of a calculated "level" with a reference or threshold value. For example, the computing device of the system can provide an indicator in the form of a word, symbol, or numerical value indicating a specific diagnosis.

The computing device may also access an output device. Exemplary output devices include, for example, a fax machine, a display, a printer, and a document. 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 that relates to a result, indication, ratio, or other factor of the method.

Furthermore, the present invention encompasses a kit adapted for performing the above-described method for predicting the risk of a female subject experiencing at least one adverse outcome associated with preeclampsia after giving birth to an infant, comprising i) a detection reagent for determining the levels of the biomarkers sFlt-1 and PlGF or ii) a detection reagent for determining the levels of the biomarkers endoglin and PlGF, or iii) a detection reagent for determining the levels of the biomarkers sFlt-1, endoglin and/or PlGF and instructions for performing the method.

As used herein, the term "kit" refers to a collection of the above-mentioned components, preferably, provided individually or in a single container. The container further comprises instructions for implementing the method of the present invention. These instructions can be in the form of a manual, or can be provided by a computer program code, which, when implemented on a computer or a data processing device, can implement the comparison mentioned in the method of the present invention, and accordingly establish a diagnosis. The computer program code can be on a data storage medium or device such as an optical storage medium (e.g., a CD), or directly on a computer or a data processing device. In addition, the kit should include at least one standard for reference as defined above, i.e., a solution of a biomarker as mentioned herein with a predefined level representing a reference level.

In some embodiments, the kit disclosed herein includes a packaged combination of at least one component or components for implementing the disclosed method. "Packaged combination" means that the kit provides a single package containing one or more components as disclosed herein, such as a combination of probes (e.g., antibodies), controls, buffers, reagents (e.g., conjugates and/or substrates) instructions, etc. A kit containing a single container is also included in the definition of "packaged combination". In some embodiments, the kit includes at least one probe, such as an antibody (having specific affinity for the epitope of a biomarker as disclosed herein). For example, the kit can include an antibody labeled with a fluorophore or an antibody as a member of a fusion protein. In the kit, the probe can be fixed and can be fixed in a specific conformation. For example, a fixed probe can be provided in the kit to specifically bind to a target protein to detect the target protein in a sample, and/or remove the target protein from the sample.

According to some embodiments, the kit includes at least one probe in at least one container, which may be fixed. The kit may also include multiple probes in one or more containers, which are optionally fixed. 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.

In some embodiments, the kit can include one or more non-fixed probes and one or more solid supports with or without fixed probes. Some such embodiments can include some or all of the reagents and supplies required for fixing the one or more probes to the solid support, or some or all of the reagents and supplies required for binding the fixed probes to a specific protein in a sample.

In certain embodiments, single probe (comprising the identical probe of multiple copies) can be fixed on single solid support, and is provided in single container.In other embodiments, two or more probes are provided in single container, and every kind of single target protein (such as specific epitope) is specific for different target proteins or different forms.In some such embodiments, fixed probe can provide (for example, with disposable form) in multiple different containers, or multiple fixed probes can provide in multiple different containers.In further embodiments, probe can be fixed on multiple different types of solid supports.Consider fixing one or more probes and any combination of one or more containers for test kit disclosed herein, and any combination thereof can be selected to obtain suitable test kit for desired use.

The container of the kit can be any container that is suitable for packaging and/or containing one or more components disclosed herein, including, for example, probes (e.g., antibodies), controls, buffers, and reagents (e.g., conjugates and/or substrates). Suitable materials include, but are not limited to, glass, plastic, cardboard or other paper products, wood, metal, and any alloys thereof. In some embodiments, the container can completely enclose the fixed one or more probes, or can simply cover the probes to minimize contamination and light exposure from dust, oils, etc. In some further embodiments, the kit can comprise a single container or multiple containers, and when multiple containers are present, each container can be the same as all other containers, different from other containers, or different from some but not all other containers.

The present invention also relates to a system for predicting the risk of a female subject experiencing at least one adverse outcome associated with preeclampsia, comprising

a) an analyzer unit configured to contact in vitro portions of a first and a second sample from a subject as described elsewhere herein with i) a reagent that specifically binds PlGF and ii) a reagent that specifically binds sFlt-1, or a reagent that specifically binds endoglin,

b) an analyzer unit configured to detect a signal from a portion of a sample from the body in contact with said reagent,

c) a computing device having a processor and in operable communication with said analysis unit, and

d) a non-transitory machine-readable medium comprising a plurality of instructions executable by the processor, which, when implemented, calculate the first and second ratios as described elsewhere herein and compare the first ratio to the second ratio to thereby predict the risk of the female subject experiencing at least one adverse outcome associated with preeclampsia.

All references mentioned above are hereby incorporated by reference with respect to their entire disclosure content as well as with respect to the specific disclosure content expressly mentioned in the above description.

Example

The following examples are intended to illustrate the present invention only and should not be construed as limiting the scope of the present invention in any way.

Example 1: Measurement of serum levels of PlGF, sFlt-1 and endoglin

Serum levels of sFlt-1, PlGF, and endoglin were determined using commercially available immunoassays. Specifically, the following assays have been used.

sFlt-1 is measured using a sandwich immunoassay using an analyzer from the Roche Elecsys™ or cobas e™ series. The assay comprises two monoclonal antibodies specific for the respective polypeptides. The first of these antibodies is biotinylated, and the second is labeled with a Tris(2,2'-bipyridyl)ruthenium(II) complex. In a first incubation step, the two antibodies are incubated with the sample. A sandwich complex is formed containing the peptide to be measured and the two different antibodies. In a next incubation step, streptavidin-coated beads are added to the complex. The beads bind to the sandwich complex. The reaction mixture is then pumped into a measuring chamber where the beads are magnetically captured on the surface of an electrode. Application of a voltage then induces chemiluminescent emission from the ruthenium complex, which is measured by a photomultiplier tube. The amount of luminescence depends on the amount of sandwich complex on the electrode. The sFlt-1 test is commercially available from Roche Diagnostics GmbH, Mannheim, Germany. Further details about the assay are provided in the package insert. The measurement range of sFlt-1 includes levels between 10 and 85,000 pg/ml.

Endoglin was measured using the Quantikine™ human endoglin/CD105 immunoassay commercially available from R&D Systems, Inc, Minneapolis, US. The assay utilizes a quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for endoglin was pre-coated onto a microplate. Standards and samples were aspirated into the wells, and any endoglin present was bound by the immobilized antibody. After washing away any unbound material, an enzyme-linked monoclonal antibody specific for endoglin was added to the wells. After washing to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells, and a color developed that was proportional to the level of endoglin bound in the initial step. Color development was stopped, and the color intensity was measured. Further details about the assay are found in the package insert. The measurement range for endoglin includes levels between 0.001 ng/L and 10 ng/ml.

PlGF was tested using two PlGF-specific antibodies in a sandwich immunoassay performed on an Elecsys™ or cobas e™ series analyzer (described above). The PlGF test was purchased from Roche Diagnostics GmbH, Mannheim, Germany. Further details about the assay are provided in the package insert. The measurement range for PlGF includes levels from 3 to 10,000 pg/ml.

Example 2: Analysis of the biomarkers sFlt-1 and PlGF in patients with consequences of 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.

Women with postpartum HELLP syndrome/postpartum preeclampsia/postpartum eclampsia

1) Women with postpartum HELLP syndrome:

sFlt-1/PlGF ratio (R1)=44

sFlt-1/PlGF ratio (R2) = 64

R2/R1= 65/44=1.45 (>=1)

2) Women with postpartum severe preeclampsia and associated liver pathology:

sFlt-1/PlGF ratio (R1)=162

sFlt-1/PlGF ratio (R2) = 283

R2/R1= 283/162=1.74 (>=1)

comparison

Women with preeclampsia (clinical onset of the disease before delivery):

1) Women with severe preeclampsia (onset and delivery during 33-36 weeks of gestation)

sFlt-1/PlGF ratio (R1)=101

sFlt-1/PlGF ratio (R2) = 19

R2/R1= 19/101=0.18 (<1)

Women without preeclampsia/eclampsia/HELLP syndrome:

1) Women with elevated liver enzymes

sFlt-1/PlGF ratio (R1)=143

sFlt-1/PlGF ratio (R2) = 73

R2/R1= 73/143=0.51 (<1)

2) Another control woman

sFlt-1/PlGF ratio (R1)=132

sFlt-1/PlGF ratio (R2) = 35

R2/R1= 35/132=0.26 (<1).

In addition, the levels of sFlt-1 and PlGF in samples obtained after delivery were compared with the levels of sFlt-1 and PlGF in samples obtained before delivery. Interestingly, in subjects with adverse outcomes associated with postpartum preeclampsia, both levels decreased after delivery. Based on the observed decreases, it is not possible to establish a risk prediction for the test patients (compared to controls) based on the levels of either single biomarker, sFlt-1 or PlGF, respectively. Therefore, the ratios disclosed herein are reliable markers for predicting the risk of adverse outcomes associated with postpartum preeclampsia.

Claims (41)

1. Use of biomarkers: i) sFlt-1 (soluble fms-like tyrosine kinase-1) or endothelial glycoprotein and ii) PlGF (placental growth factor), or reagents that specifically bind said biomarkers, in a preparation apparatus for a method of predicting the risk of at least one preeclampsia-related adverse outcome in a female subject after childbirth, said method comprising the following steps a) Measurements were taken from the first sample obtained from a female subject with a safe pregnancy before delivery of the infant. i) The level of the biomarker sFlt-1 or the biomarker endothelial glycoprotein, and ii) The level of the biomarker PlGF, b) Calculate the first ratio of the levels of the biomarkers measured in step a). c) After childbirth, the levels of the biomarkers as measured in step a) are obtained from a second sample of the female subject. d) Calculate the second ratio of the levels measured in step c), and e) Compare the second ratio with the first ratio. 2. The use of claim 1, wherein in steps a) and c), the levels of biomarkers sFlt-1 and PlGF are measured. 3. The use of claim 2, wherein at least one preeclampsia-related adverse outcome is selected from postpartum preeclampsia, postpartum eclampsia, and postpartum HELLP syndrome. 4. Use according to any one of claims 1 to 3, wherein the female subject with a safe pregnancy does not exhibit adverse consequences related to preeclampsia before giving birth to the infant. 5. Use according to any one of claims 1 to 3, wherein the female subject who has a safe pregnancy does not have preeclampsia before giving birth to the infant. 6. Use according to any one of claims 1 to 3, wherein the female subject who has a safe pregnancy does not have eclampsia and/or HELLP syndrome before giving birth to the infant. 7. Use according to any one of claims 1 to 3, wherein the first sample was obtained within 48 hours prior to delivery of the infant. 8. Use according to any one of claims 1 to 3, wherein the first sample was obtained within 24 hours prior to the delivery of the infant. 9. Use according to any one of claims 1 to 3, wherein the second sample was obtained within 24 hours after the delivery of the infant. 10. Use according to any one of claims 1 to 3, wherein the second sample was obtained within 16 hours after the delivery of the infant. 11. The use of any one of claims 1 to 3, wherein the first and second ratios are the ratio of sFlt-1 to PlGF or the ratio of endothelial glycoprotein to PlGF, and wherein an increase in the second ratio or a substantially unchanged second ratio compared to the first ratio indicates a subject at risk of developing at least one preeclampsia-related adverse outcome after childbirth, and/or wherein a decrease in the second ratio compared to the first ratio indicates a subject not at risk of developing preeclampsia-related adverse outcomes after childbirth. 12. The use of any one of claims 1 to 3, wherein the first and second ratios are the ratio of PlGF to sFlt-1 or the ratio of PlGF to endothelial glycoprotein, and wherein a decrease in the second ratio or a substantially unchanged second ratio compared to the first ratio indicates a subject at risk of developing at least one preeclampsia-related adverse outcome after childbirth, and/or an increase in the second ratio compared to the first ratio indicates a subject not at risk of developing preeclampsia-related adverse outcomes after childbirth. 13. The use of any one of claims 1 to 3, wherein the subject is a person. 14. Use according to any one of claims 1 to 3, wherein the sample is a blood, serum, or plasma sample, or wherein the sample is a urine sample. 15. Use according to any one of claims 1 to 3, wherein the risk of at least one preeclampsia-related adverse outcome is predicted within seven days after the delivery of the infant. 16. Use according to any one of claims 1 to 3, wherein the risk of at least one preeclampsia-related adverse outcome is predicted within 72 hours after the delivery of the infant. 17. Use according to any one of claims 1 to 3, wherein the risk of at least one preeclampsia-related adverse outcome is predicted within 48 hours after the delivery of the infant. 18.i) Biomarkers sFlt-1 and PlGF, ii) Reagents that specifically bind to sFlt-1 and reagents that specifically bind to PlGF, iii) Biomarkers endothelial glycoprotein and PlGF, and/or iv) Reagents that specifically bind to endothelial glycoproteins and reagents that specifically bind to PlGF. Use in a preparation apparatus for predicting the risk of at least one preeclampsia-related adverse outcome in a woman subject before delivery of a baby, in a first sample obtained from a woman subject with a safe pregnancy and in a second sample obtained from the woman subject after delivery of a baby. 19. The use of claim 18, wherein the use further comprises calculating a first and a second ratio of sFlt-1 or endothelial glycoprotein and PlGF, and comparing the first ratio with the second ratio. 20. The use of claim 18 or 19, wherein said reagent is an antibody. 21. A device adapted for predicting the risk of at least one preeclampsia-related adverse outcome following childbirth in a female subject, said device comprising: a) An analyzer unit comprising reagents specifically binding sFlt-1 and/or endothelial glycoprotein and reagents specifically binding PlGF, said unit being adapted to measure the levels of sFlt-1 and/or endothelial glycoprotein and PlGF in a first sample of a female subject obtained before delivery and a second sample of the female subject obtained after delivery; and b) An evaluation unit, which includes a data processor that has been implemented with algorithms for carrying out the following steps: i) Calculate a first ratio from the levels of sFlt-1 or endothelial glycoprotein and PlGF measured in the first sample, and calculate a second ratio from the levels of sFlt-1 or endothelial glycoprotein and PlGF measured in the second sample; ii) Compare the values of the first and second ratios, and iii) Predict the risk of at least one preeclampsia-related adverse outcome following the delivery of the infant by the subject. 22. Use of biomarkers: i) sFlt-1 (soluble fms-like tyrosine kinase-1) or endothelial glycoprotein and ii) PlGF (placental growth factor), or reagents that specifically bind said biomarkers, in the preparation of a kit for a method of predicting the risk of at least one preeclampsia-related adverse outcome in a female subject after childbirth, said method comprising the following steps a) Measurements were taken from the first sample obtained from a female subject with a safe pregnancy before delivery of the infant. i) The level of the biomarker sFlt-1 or the biomarker endothelial glycoprotein, and ii) The level of the biomarker PlGF, b) Calculate the first ratio of the levels of the biomarkers measured in step a). c) After childbirth, the levels of the biomarkers as measured in step a) are obtained from a second sample of the female subject. d) Calculate the second ratio of the levels measured in step c), and e) Compare the second ratio with the first ratio. 23. The use of claim 22, wherein in steps a) and c), the levels of biomarkers sFlt-1 and PlGF are measured. 24. The use of claim 22, wherein at least one preeclampsia-related adverse outcome is selected from postpartum preeclampsia, postpartum eclampsia, and postpartum HELLP syndrome. 25. Use according to any one of claims 22 to 24, wherein the female subject with a healthy pregnancy does not exhibit adverse consequences related to preeclampsia before giving birth to the infant. 26. Use according to any one of claims 22 to 24, wherein the female subject with a safe pregnancy does not have preeclampsia before giving birth to the infant. 27. Use according to any one of claims 22 to 24, wherein the female subject who has a safe pregnancy does not have eclampsia and/or HELLP syndrome before giving birth to the infant. 28. Use according to any one of claims 22 to 24, wherein the first sample was obtained within 48 hours prior to delivery of the infant. 29. Use according to any one of claims 22 to 24, wherein the first sample was obtained within 24 hours prior to delivery of the infant. 30. Use according to any one of claims 22 to 24, wherein the second sample has been obtained within 24 hours after the delivery of the infant. 31. Use according to any one of claims 22 to 24, wherein the second sample has been obtained within 16 hours after the delivery of the infant. 32. The use of any one of claims 22 to 24, wherein the first and second ratios are the ratio of sFlt-1 to PlGF or the ratio of endothelial glycoprotein to PlGF, and wherein an increase in the second ratio or a substantially unchanged second ratio compared to the first ratio indicates a subject at risk of developing at least one preeclampsia-related adverse outcome after childbirth, and/or wherein a decrease in the second ratio compared to the first ratio indicates a subject not at risk of developing preeclampsia-related adverse outcomes after childbirth. 33. The use of any one of claims 22 to 24, wherein the first and second ratios are the ratio of PlGF to sFlt-1 or the ratio of PlGF to endothelial glycoprotein, and wherein a decrease in the second ratio or a substantially unchanged second ratio compared to the first ratio indicates a subject at risk of developing at least one preeclampsia-related adverse outcome after childbirth, and/or an increase in the second ratio compared to the first ratio indicates a subject not at risk of developing preeclampsia-related adverse outcomes after childbirth. 34. The use of any one of claims 22 to 24, wherein the subject is a person. 35. The use of any one of claims 22 to 24, wherein the sample is a blood, serum, or plasma sample, or wherein the sample is a urine sample. 36. The use of any one of claims 22 to 24, wherein the risk of at least one preeclampsia-related adverse outcome is predicted within seven days after the delivery of the infant. 37. The use of any one of claims 22 to 24, wherein the risk of at least one preeclampsia-related adverse outcome is predicted within 72 hours after the delivery of the infant. 38. The use of any one of claims 22 to 24, wherein the risk of at least one preeclampsia-related adverse outcome is predicted within 48 hours after the delivery of the infant. 39.i) Biomarkers sFlt-1 and PlGF, ii) Reagents that specifically bind to sFlt-1 and reagents that specifically bind to PlGF, iii) Biomarkers endothelial glycoprotein and PlGF, and/or iv) Reagents that specifically bind to endothelial glycoproteins and reagents that specifically bind to PlGF. The use of a kit in the preparation of a kit obtained from a first sample obtained from a female subject with a safe pregnancy before delivery of a baby and from a second sample obtained from the same female subject after delivery of a baby, the kit being used to predict the risk of at least one preeclampsia-related adverse outcome in the female subject after delivery of a baby. 40. The use of claim 39, wherein the use further comprises calculating a first and a second ratio of sFlt-1 or endothelial glycoprotein and PlGF, and comparing the first ratio with the second ratio. 41. The use of claim 39 or 40, wherein the reagent is an antibody.