WO2022186821A1

WO2022186821A1 - Methods and compositions for providing a preeclampsia assessment using leptin and ceramide

Info

Publication number: WO2022186821A1
Application number: PCT/US2021/020417
Authority: WO
Inventors: Bruce Xuefeng Ling; Limin Chen; Harvey J. COHEN; Shiying Hao; Doff B. Mcelhinney; Karl G. SYLVESTER; Lu Tian; Ronald J. WONG; Gary M. SHAW; David K. Stevenson
Original assignee: Mprobe Inc.,; The Board Of Trustees Of The Leland Stanford Junior
Priority date: 2021-03-02
Filing date: 2021-03-02
Publication date: 2022-09-09

Abstract

Preeclampsia markers, preeclampsia marker panels, and methods for obtaining a preeclampsia marker level representation for a sample are provided. These composition and methods find use in a number of applications, including, for example, diagnosing preeclampsia, prognosing preeclampsia, monitoring a subject with preeclampsia, and determining a treatment for preeclampsia. In addition, systems, devices, and kits thereof that find use in practicing the subject methods are provided.

Description

METHODS AND COMPOSITIONS FOR PROVIDING A PREECLAMPSIA ASSESSMENT

USING LEPTIN AND CERAMIDE

FIELD OF THE INVENTION

[0001] This disclosure of provisional patent application pertains to methods and compositions for providing a preeclampsia assessment using Leptin and ceramides.

BACKGROUND OF THE INVENTION

[0002] Preeclampsia is a serious multisystem complication of pregnancy with adverse effects for mothers and babies. The incidence of the disorder is around 5-8% of all pregnancies in the U.S. and worldwide, and the disorder is responsible for 18% of all maternal deaths in the U.S. The causes and pathogenesis of preeclampsia remain uncertain, and the diagnosis relies on nonspecific laboratory and clinical signs and symptoms that occur late in the disease process, sometimes making the diagnosis and clinical management decisions difficult. Earlier and more reliable disease diagnosing, prognosing and monitoring will lead to more timely and personalized preeclampsia treatments and significantly advance our understanding of preeclampsia pathogenesis.

SUMMARY OF THE INVENTION

[0003] Preeclampsia markers, preeclampsia marker panels, and methods for obtaining a preeciampsia marker level representation for a sample are provided. These compositions and methods find use in a number of applications, including, for example, diagnosing preeclampsia, prognosing a preeclampsia, monitoring a subject with preeclampsia, and determining a treatment for preeclampsia. In addition, systems, devices and kits thereof that find use in practicing the subject methods are provided.

[0004] In some aspects of the invention, a panel of preeclampsia markers is provided, the panel comprising one or more preeciampsia markers selected from the group consisting of Leptin (LEP), Ceramide (d18:1/25:0), and Ceramide (d18:1/26:0).

[0005] In some aspects of the invention, a method is provided for providing a preeclampsia marker level representation for a subject. In some embodiments, the method comprises evaluating a panel of preeclampsia markers in a blood sample from a subject to determine the level of each preeclampsia marker in the blood sample; and obtaining the preeclampsia marker level representation based on the level of each preeclampsia marker in the panel. In some embodiments, the panel comprises Leptin (LEP). In some embodiments, the panel comprises Leptin (LEP) and Ceramide (d 18:1/25:0). In some embodiments, the panel comprises Leptin (LEP) and Ceramide (d18:1/26:0). In some embodiments, the panel comprises one or more preeclampsia markers selected from the group consisting of Leptin (LEP), Ceramide (d18: 1/25:0), Ceramide (d 18: 1/26:0). In some embodiments, the method further comprises providing a report of the preeclampsia marker level representation. In certain embodiments, the preeclampsia marker representation is a preeclampsia score.

[0006] In some aspects of the invention, a method is provided for providing a preeclampsia assessment for a subject. In some embodiments, the preeclampsia assessment is a diagnosis of preeclampsia. In some embodiments, the method comprises obtaining a preeclampsia marker level representation for a sample from a subject, e.g. as described above or elsewhere herein, and providing a preeclampsia diagnosis for the subject based on the preeclampsia marker level representation. In some embodiments, the method further comprises comparing the preeclampsia marker level representation to a preeclampsia phenotype determination element, and providing a preeclampsia diagnosis for the subject based on the comparison. In some embodiments, the subject has symptoms of preeclampsia. In other embodiments, the subject is asymptomatic for preeclampsia. In some embodiments, the subject has one or more risk factors associated with preeclampsia. In other embodiments, the subject has no risk factors associated with preeclampsia.

[0007] The methods may be particularly suitable for certain pregnant women, such as those that have history of preeclampsia, have obesity, have babies less than two years or more than 10 years apart, are older than 40, have history of certain conditions including chronic high blood pressure, migraine headaches, type 1 or type 2 diabetes, kidney disease, a tendency to develop blood clots, or lupus.

[0008] Once the diagnosis of preeclampsia is determined, the woman can be subject to a procedure that helps ameliorate the preeclampsia. Examples of such procedures include, without limitation, medications to lower blood pressure, use of corticosteroids, anticonvulsant medication such as magnesium sulfate, bed rest, and consideration of delivery if the diagnosis was made at or after 37 gestational weeks.

[0009] In some aspects of the invention, a kit is provided for making a preeclampsia assessment for a sample. In some embodiments, the preeclampsia assessment is a preeclampsia diagnosis. In some embodiments, the kit comprises one or more detection elements for measuring the amount of marker in a sample for a panel of preeclampsia markers comprising Leptin (LEP). In some embodiments, the kit comprises one or more detection elements for measuring the amount of marker in a sample for a panel of preeclampsia markers comprising Leptin (LEP), Ceramide (d18:1/25:0), Ceramide (d 18: 1/26:0). In some embodiments, the kit further comprises a preeclampsia phenotype determination element. In some embodiments, the one or more detection elements detect the level of marker polypeptides in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. The patent or application file contains at least one drawing. Copies of this patent or patent application publication with drawing(s) will be provided by the Office upon request and payment of the necessary fee. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0011] Figure 1. Study outline of discovery and testing of PE biomarkers.

[0012] Figure 2. Serial blood sampling from each normal term and PE subject at different GAs. Times of sample collections, infant deliveries, and confirmatory PE diagnoses of individual women (denoted by each row) are represented by black circles, black squares, and red-filled triangles, respectively.

[0013] Figure 3. Concentrations in maternal serum of LEP, Ceramide (d 18: 1/25:0), and Ceramide (d 18: 1/26:0) as a function of gestational age at blood draw in the discovery cohort. Loess smooth lines were plotted for PE and controls, respectively.

[0014] Figure 4. Concentration ratio of LEP/Ceramide (d 18: 1 /25:0) (Left) and LEP/Ceramide (d18:1/25:0) (Right) in maternal serum as a function of gestational age at blood draw in the discovery cohort. Loess smooth lines were plotted for PE and controls, respectively.

[0015] Figure 5. Concentrations in maternal serum of LEP, Ceramide (d18:1/25:0), and Ceramide (d 18: 1/26:0) as a function of gestational age at blood draw in the testing cohort. Loess smooth lines were plotted for PE and controls, respectively.

[0016] Figure 6. Top: Concentration ratio of LEP/Ceramide (d18:1/25:0) (Left) and LEP/Ceramide (d18:1/25:0) (Right) in maternal serum as a function of gestational age at blood draw in the discovery cohort. LEP was normalized by BMI prior to pregnancy. Loess smooth lines were plotted for PE and controls, respectively. Bottom: ROCAUC in different GA windows of LEP/Ceramide (d18:1/25:0) ratio, LEP/Ceramide (d18:1/26:0) ratio, and sFlt-1/PIGF in differentiating impending PE from normal. 25-0 Cer: Ceramide (d18:1/25:0). 25-0 Cer: Ceramide (d18: 1/26:0).

[0017] Figure 7. Time-to-event analysis of the LEP/Ceramide (d 18: 1 /25:0) ratio and sFlt-1/PIGF ratio in the testing cohort. LEP was normalized by BMI prior to pregnancy. X axis represents the time to confirmative diagnosis of PE, and Y axis represents % of patients identified by the marker as having impending PE.

[0018] Figure 8. Sensitivity, specificity, PPV, and NPV of the LEP/Ceramide

(d 18: 1/25:0) ratio and the LEP/Ceramide (d18:1/26:0) ratio of identifying impending PE in the testing cohort. Values were calculated at 5-29 wks and 12-20 wks, respectively.

DETAILED DESCRIPTION OF THE INVENTION

[0019] Preeclampsia markers, preeclampsia marker panels, and methods for obtaining a preeclampsia marker level representation for a sample are provided. These compositions and methods find use in a number of applications, including, for example, diagnosing preeclampsia, prognosing a preeclampsia, monitoring a subject with preeclampsia, and determining a treatment for preeclampsia. In addition, systems, devices and kits thereof that find use in practicing the subject methods are provided. These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the compositions and methods as more fully described below.

[0020] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0021] Unless defined otherwise, ail technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0022] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. [0023] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.

[0024] As summarized above, aspects of the subject invention include methods, compositions, systems and kits that find use in providing a preeclampsia assessment, e.g. diagnosing, prognosing, monitoring, and/or treating preeclampsia in a subject. By "preeclampsia" or "pre-eclampsia" it is meant a multisystem complication of pregnancy that may be accompanied by one or more of high blood pressure, proteinuria, swelling of the hands and face/eyes (edema), sudden weight gain, higher-than-normai liver enzymes, and thrombocytopenia. Preeclampsia typically occurs in the third trimester of pregnancy, but in severe cases, the disorder occurs in the second trimester, e.g., after about the 22^nd week of pregnancy. If unaddressed, preeclampsia can lead to eclampsia, i.e. seizures that are not related to a preexisting brain condition. By "diagnosing" a preeclampsia or "providing a preeclampsia diagnosis," it is generally meant providing a preeclampsia determination, e.g. a determination as to whether a subject (e.g. a subject that has clinical symptoms of preeclampsia, a subject that is asymptomatic for preeclampsia but has risk factors associated with preeclampsia, a subject that is asymptomatic for preeclampsia and has no risk factors associated with preeclampsia) is presently affected by preeclampsia; a classification of the subject’s preeclampsia into a subtype of the disease or disorder; a determination of the severity of preeclampsia; and the like. By “prognosing" a preeclampsia, or "providing a preeclampsia prognosis," it is generally meant providing a preeclampsia prediction, e.g. a prediction of a subject's susceptibility, or risk, of developing preeclampsia; a prediction of the course of disease progression and/or disease outcome, e.g. expected onset of the preeclampsia, expected duration of the preeclampsia, expectations as to whether the preeclampsia will develop into eclampsia, etc.; a prediction of a subject's responsiveness to treatment for the preeclampsia, e.g., positive response, a negative response, no response at all; and the like. By "monitoring" a preeciampsia, it is generally meant monitoring a subject's condition, e.g. to inform a preeclampsia diagnosis, to inform a preeclampsia prognosis, to provide information as to the effect or efficacy of a preeclampsia treatment, and the like. By "treating" a preeciampsia it is meant prescribing or providing any treatment of a preeclampsia in a mammal, and indudes: (a) preventing the preeclampsia from occurring in a subject which may be predisposed to preeclampsia but has not yet been diagnosed as having it; (b) inhibiting the preeclampsia, i.e., arresting its development; or (c) relieving the preeclampsia, i.e., causing regression of the preeclampsia. [0025] In describing the subject invention, compositions useful for providing a preeclampsia assessment will be described first, followed by methods, systems and kits for their use.

PREECLAMPSIA MARKERS AND PANELS

[0026] In some aspects of the invention, preeclampsia markers and panels of preeclampsia markers are provided. By a "preeclampsia marker" it is meant a molecular entity whose representation in a sample is associated with a preeclampsia phenotype. For example, a preeclampsia marker may be differentially represented, i.e. represented at a different level, in a sample from an individual that will develop or has developed preeclampsia as compared to a healthy individual. In some instances, an elevated level of marker is associated with the preeclampsia phenotype. For example, the concentration of marker in a sample may be 1.5- fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 7.5-fold, 10-fold, or greater in a sample assodated with the preeclampsia phenotype than in a sample not associated with the preeclampsia phenotype. In other instances, a reduced level of marker is assodated with the preeclampsia phenotype. For example, the concentration of marker in a sample may be 10% less, 20% less, 30% less, 40% less, 50% less or more in a sample associated with the preeclampsia phenotype than in a sample not associated with the preeclampsia phenotype.

[0027] Preeclampsia markers may indude proteins assodated with preeclampsia and their corresponding genetic sequences, i.e. mRNA, DNA, etc. By a "gene" or "recombinant gene" it is meant a nucleic acid comprising an open reading frame that encodes for the protein.

[0028] The boundaries of a coding sequence are determined by a start codon at the 5" (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A transcription termination sequence may be located 3' to the coding sequence. In addition, a gene may optionally include its natural promoter (i.e., the promoter with which the exons and introns of the gene are operably linked in a non-recombinant cell, i.e., a naturally occurring cell), and associated regulatory sequences, and may or may not have sequences upstream of the AUG start site, and may or may not indude untranslated leader sequences, signal sequences, downstream untranslated sequences, transcriptional start and stop sequences, polyadenylation signals, translational start and stop sequences, ribosome binding sites, and the like.

[0029] As demonstrated in the examples of the present disdosure, the inventors have identified a number of molecular entities that are assodated with preeclampsia and that find use in combination (i.e. as a panel) in providing a preeclampsia assessment, e.g. diagnosing preeclampsia, prognosing a preeclampsia, monitoring a subject with preeclampsia, determining a treatment for a subject affected with preeclampsia, and the like. These include, but are not limited to, Leptin (LEP), Ceramide (d18:1/25:0), and Ceramide (d18:1/26:0).

[0030] As mentioned above, also provided herein are preeclampsia panels. By a “panel” of preeclampsia markers it is meant two or more preeclampsia markers, e.g. 2 or more, 3 or more, or 4 or more markers, whose levels, when considered in combination, find use in providing a preeclampsia assessment, e.g. making a preeclampsia diagnosis, prognosis, monitoring, and/or treatinent. Of particular interest are panels that comprise the preeclampsia marker Leptin (LEP), Ceramide (d18:1/25:0), and Ceramide (d18:1/26:0). For example, in some embodiments, the preeclampsia panel may comprise LEP and Ceramide (d18:1/25:0), or LEP and Ceramide (d18:1/26:0).

[0031] Other combinations of preeclampsia markers that find use as preeclampsia panels in the subject methods may be readily identified by the ordinarily skilled artisan using any convenient statistical methodology, e.g. as known in the art or described in the working examples herein. For example, the panel of analytes may be selected by combining genetic algorithm (GA) and all paired (AP) support vector machine (SVM) methods for preeclampsia classification analysis. Predictive features are automatically determined, e.g. through iterative GA/SVM, leading to very compact sets of non- redundant preeclampsia-relevant analytes with the optimal classification performance. While different classifier sets will typically harbor only modest overlapping gene features, they will have similar levels of accuracy in providing a preeclampsia assessment to those described above and in the working examples herein

METHODS

[0032] In some aspects of the invention, methods are provided for obtaining a preeclampsia marker level representation for a subject. By a preeclampsia marker level representation, it is meant a representation of the levels of one or more of the subject preeclampsia marker(s), e.g. a panel of preeclampsia markers, in a biological sample from a subject. The term "biological sample" encompasses a variety of sample types obtained from an organism and can be used in a diagnostic, prognostic, or monitoring assay. The term encompasses blood and other liquid samples of biological origin or cells derived therefrom and the progeny thereof. The term encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components. The term encompasses a clinical sample, and also includes cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples. Clinical samples for use in the methods of the invention may be obtained from a variety of sources, particularly blood samples.

[0033] Sample sources of particular interest indude blood samples or preparations thereof, e.g., whole blood, or serum or plasma, and urine. In many embodiments, a suitable initial source for the human sample is a blood sample. As such, the sample employed in the subject assays is generally a blood-derived sample. The blood derived sample may be derived from whole blood or a fraction thereof, e.g., serum, plasma, etc., where in some embodiments the sample is derived from blood, allowed to clot, and the serum separated and collected to be used to assay.

[0034] In some embodiments the sample is a serum or serum-derived sample. Any convenient methodology for producing a fluid serum sample may be employed. In many embodiments, the method employs drawing venous blood by skin puncture (e.g., finger stick, venipuncture) into a clotting or serum separator tube, allowing the blood to clot, and centrifuging the serum away from the dotted blood. The serum is then collected and stored until assayed. Once the patient derived sample is obtained, the sample is assayed to determine the level of preeclampsia marker(s).

[0035] The subject sample is typically obtained from the individual during the first, second or third trimester of gestation. By "gestation” it is meant the duration of pregnancy in a mammal, i.e. the time interval of development from fertilization until birth, plus two weeks, i.e. to the first day of the last menstrual period. By the first, second, or third trimester, it is meant the first, second, or third portions of gestation, each segment being 3 months long. Thus, for example, by the "first trimester" is meant from the first day of the last menstrual period through the 13th week of gestation; by the "second trimester" it is meant from the 14th through 27th week of gestation; and by the "third trimester" it is meant from the 28th week through birth, i.e. 38 - 42 weeks of gestation.

[0036] Once a sample is obtained, it can be used directly, frozen, or maintained in appropriate culture medium for short periods of time. Typically the samples will be from human patients, although animal models may find use, e.g. equine, bovine, porcine, canine, feline, rodent, e.g. mice, rats, hamster, primate, etc. Any convenient tissue sample that demonstrates the differential representation in a patient with preeclampsia of the one or more preeclampsia markers disclosed herein may be evaluated in the subject methods. Typically, a suitable sample source will be derived from fluids into which the molecular entity of interest, i.e. the RNA transcript or protein, has been released.

[0037] The subject sample may be treated in a variety of ways so as to enhance detection of the one or more preeclampsia markers. For example, where the sample is blood, the red blood cells may be removed from the sample (e.g., by centrifugation) prior to assaying. Such a treatment may serve to reduce the non-specific background levels of detecting the level of a preeclampsia marker using an affinity reagent. Detection of a preeclampsia marker may also be enhanced by concentrating the sample using procedures well known in the art (e.g. acid precipitation, alcohol precipitation, salt precipitation, hydrophobic precipitation, filtration. In some embodiments, the pH of the test and control samples will be adjusted to, and maintained at, a pH which approximates neutrality. Such a pH adjustment will prevent complex formation, thereby providing a more accurate quantitation of the level of marker in the sample. In embodiments where the sample is urine, the pH of the sample is adjusted and the sample is concentrated in order to enhance the detection of the marker.

[0038] In practicing the subject methods, the level(s) of preeclampsia markers) in the biological sample from an individual are evaluated. The level of one or more preeclampsia markers in the subject sample may be evaluated by any convenient method. For example, preeclampsia gene expression levels may be detected by measuring the levels/amounts of one or more nucleic acid transcripts, e.g. mRNAs, of one or more preeclampsia genes. Protein markers may be detected by measuring the levels/amounts of one or more proteins/polypeptides. The terms “evaluating”, “assaying", “measuring", “assessing," and "determining" are used interchangeably to refer to any form of measurement, including determining if an element is present or not, and including both quantitative and qualitative determinations. Evaluating may be relative or absolute.

[0039] For example, the level of at least one preeclampsia marker may be evaluated by detecting in a sample the amount or level of one or more proteins/polypeptides or fragments thereof to arrive at a protein level representation. The terms "protein" and "polypeptide" as used in this application are interchangeable. "Polypeptide" refers to a polymer of amino acids (amino acid sequence) and does not refer to a specific length of the molecule. Thus peptides and oligopeptides are included within the definition of polypeptide. This terni also refers to or includes post-translationally modified polypeptides, for example, glycosylated polypeptide, acetylated polypeptide, phosphorylated polypeptide and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid, polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.

[0040] When protein levels are to be detected, any convenient protocol for evaluating protein levels may be employed wherein the level of one or more proteins in the assayed sample is determined. For example, one representative and convenient type of protocol for assaying protein levels is ELISA. In ELISA and ELISA-based assays, one or more antibodies specific for the proteins of interest may be immobilized onto a selected solid surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, the assay plate wells are coated with a non-specific "blocking" protein that is known to be antigenically neutral with regard to the test sample such as bovine serum albumin (BSA), casein or solutions of powdered milk. This allows for blocking of non-specific adsorption sites on the immobilizing surface, thereby reducing the background caused by non-specific binding of antigen onto the surface. After washing to remove unbound blocking protein, the immobilizing surface is contacted with the sample to be tested under conditions that are conducive to immune complex (antigen/antibody) formation. Such conditions include diluting the sample with diluents such as BSA or bovine gamma globulin (BGG) in phosphate buffered saline (PBS)ZTweenor PBSATriton-X 100, which also tend to assist in the reduction of nonspecific background, and allowing the sample to incubate for about 2-4 hrs at temperatures on the order of about 25°-27’C (although other temperatures may be used). Following incubation, the antisera-contacted surface is washed so as to remove non- immunocomplexed material. An exemplary washing procedure includes washing with a solution such as PBS/Tween, PBS/Triton-X 100, or borate buffer. The occurrence and amount of immunocomplex formation may then be determined by subjecting the bound immunocomplexes to a second antibody having specificity for the target that differs from the first antibody and detecting binding of the second antibody. In certain embodiments, the second antibody will have an associated enzyme, e.g. urease, peroxidase, or alkaline phosphatase, which will generate a color precipitate upon incubating with an appropriate chromogenic subskate. For example, a urease or peroxidase-conjugated anti-human IgG may be employed, for a period of time and under conditions which favor the development of immunocomplex formation (e.g., incubation for 2 hrs at room temperature in a PBS-containing solution such as PBS/Tween). After such incubation with the second antibody and washing to remove unbound material, the amount of label is quantified, for example by incubation with a chromogenic substrate such as urea and bromocresol purple in the case of a urease label or 2,2'-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H₂O₂, in the case of a peroxidase label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectrum spectrophotometer.

[0041] The preceding format may be altered by first binding the sample to the assay plate. Then, primary antibody is incubated with the assay plate, followed by detecting of bound primary antibody using a labeled second antibody with spedficity for the primary antibody.

[0042] The solid substrate upon which the antibody or antibodies are immobilized can be made of a wide variety of materials and in a wide variety of shapes, e.g., microtiter plate, microbead, dipstick, resin partide, etc. The substrate may be chosen to maximize signal to noise ratios, to minimize background binding, as well as for ease of separation and cost. Washes may be effected in a manner most appropriate for the substrate being used, for example, by removing a bead or dipstick from a reservoir, emptying or diluting a reservoir such as a microtiter plate well, or rinsing a bead, partide, chromatograpic column or filter with a wash solution or solvent.

[0043] Alternatively, non-ELISA based-methods for measuring the levels of one or more proteins in a sample may be employed. Representative examples indude but are not limited to mass spectrometry, proteomic arrays, xMAP™ microsphere technology, flow cytometry, western blotting, and immunohistochemistry.

[0044] As another example, the level of at least one preeclampsia marker may be evaluated by deteding in a patient sample the amount or level of one or more RNA transcripts or a fragment thereof encoded by the gene of interest to arrive at a nucleic acid marker representation. The level of nucleic acids in the sample may be deteded using any convenient protocol. While a variety of different manners of deteding nucleic adds are known, such as those employed in the field of differential gene expression analysis, one representative and convenient type of protocol for generating marker representations is array-based gene expression profiling protocols. Such applications are hybridization assays in which a nucleic acid that displays "probe" nudeic acids for each of the genes to be assayed/profiled in the marker representation to be generated is employed. In these assays, a sample of target nudeic acids is first prepared from the initial nucleic add sample being assayed, where preparation may include labeling of the target nudeic adds with a label, e.g., a member of signal produdng system. Following target nucleic acid sample preparation, the sample is contacted with the array under hybridization conditions, whereby complexes are formed between target nucleic adds that are complementary to probe sequences attached to the array surface. The presence of hybridized complexes is then detected, either qualitatively or quantitatively.

[0045] Specific hybridization technology which may be practiced to generate the marker representations employed in the subject methods includes the technology described in U.S. Patent Nos.: 5,143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806; 5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661,028; 5,800,992; the disdosures of which are herein incorporated by reference; as well as WO 95/21265; WO 96/31622; WO 97/10365; WO 97/27317; EP 373203; and EP 785280. In these methods, an array of "probe" nudeic adds that indudes a probe for each of the phenotype determinative genes whose expression is being assayed is contacted with target nudeic adds as described above. Contact is carried out under hybridization conditions, e.g., stringent hybridization conditions, and unbound nucleic acid is then removed. The term "stringent assay conditions" as used herein refers to conditions that are compatible to produce binding pairs of nucleic adds, e.g., surface bound and solution phase nucleic acids, of sufficient complementarity to provide for the desired level of specifidty in the assay while being less compatible to the formation of binding pairs between binding members of insufficient complementarity to provide for the desired specifidty. Stringent assay conditions are the summation or combination (totality) of both hybridization and wash conditions.

[0046] The resultant pattern of hybridized nudeic add provides information regarding expression for each of the genes that have been probed, where the expression information is in terms of whether or not the gene is expressed and, typically, at what level, where the expression data, i.e., marker representation (e.g., in the form of a transcriptosome), may be both qualitative and quantitative.

[0047] Alternatively, non-array based methods for quantitating the level of one or more nucleic acids in a sample may be employed, including those based on amplification protocols, e.g., Polymerase Chain Reaction (PCR)-based assays, including quantitative PCR, reverse-transcription PCR (RT-PCR), real-time PCR, and the like.

[0048] The resultant data provides information regarding levels in the sample for each of the markers that have been probed, wherein the information is in terms of whether or not the marker is present and, typically, at what level, and wherein the data may be both qualitative and quantitative. As such, where detection is qualitative, the methods provide a reading or evaluation, e.g., assessment, of whether or not the target marker, e.g., nucleic add or protein, is present in the sample being assayed. In yet other embodiments, the methods provide a quantitative detection of whether the target marker is present in the sample being assayed, i.e., an evaluation or assessment of the actual amount or relative abundance of the target analyte, e.g., nucleic acid or protein in the sample being assayed. In such embodiments, the quantitative detection may be absolute or, if the method is a method of detecting two or more different analytes, e.g., target nucleic acids or protein, in a sample, relative. As such, the term "quantifying" when used in the context of quantifying a target analyte, e.g., nucleic acid(s) or protein(s), in a sample can refer to absolute or to relative quantification. Absolute quantification may be accomplished by inclusion of known concentrations ) of one or more control analytes and referencing the detected level of the target analyte with the known control analytes (e.g., through generation of a standard curve). Alternatively, relative quantification can be accomplished by comparison of detected levels or amounts between two or more different target analytes to provide a relative quantification of each of the two or more different analytes, e.g., relative to each other. [0049] Once the level of the one or more preeclampsia markers has been determined, the measurements) may be analyzed in any of a number of ways to obtain a preeclampsia marker level representation.

[0050] For example, the measurements of the one or more preeclampsia markers may be analyzed individually to develop a preeclampsia profile. As used herein, a “preeclampsia profile" is the normalized level of one or more preeclampsia markers in a patient sample, for example, the normalized level of serological protein concentrations in a patient sample. A profile may be generated by any of a number of methods known in the art. For example, the level of each marker may be log₂ transformed and normalized relative to the expression of a selected housekeeping gene, or relative to the signal across a whole panel, etc. Other methods of calculating a preeclampsia profile will be readily known to the ordinarily skilled artisan.

[0051] As another example, the measurements of a panel of preeclampsia markers may be analyzed collectively to arrive at a single preeclampsia score. By a ‘preeclampsia score" it is meant a single metric value that represents the weighted levels of each of the preeclampsia markers in the preeclampsia panel. As such, in some embodiments, the subject method comprises detecting the level of markers of a preeclampsia panel in the sample, and calculating a preeclampsia score based on the weighted levels of the preeclampsia markers. A preeclampsia score for a patient sample may be calculated by any of a number of methods and algorithms known in the art for calculating biomarker scores. For example, weighted marker levels, e.g. log₂ transformed and normalized marker levels that have been weighted by, e.g., multiplying each normalized marker level to a weighting factor, may be totaled and in some cases averaged to arrive at a single value representative of the panel of preeclampsia markers analyzed.

[0052] In some instances, the weighting factor, or simply "weight" for each marker in a panel may be a reflection of the change in analyte level in the sample. For example, the analyte level of each preeclampsia marker may be log transformed and weighted either as 1 (for those markers that are increased in level in preeclampsia) or -1 (for those markers that are decreased in level in preeclampsia), and the ratio between the sum of increased markers as compared to decreased markers determined to arrive at a preeclampsia signature. In other instances, the weights may be reflective of the importance of each marker to the specifidty, sensitivity and/or accuracy of the marker panel in making the diagnostic, prognostic, or monitoring assessment. Such weights may be determined by any convenient statistical machine learning methodology, e.g. Principle Component Analysis (PCA), linear regression, support vector machines (SVMs), and/or random forests of the dataset from which the sample was obtained may be used. In some instances, weights for each marker are defined by the dataset from which the patient sample was obtained. In other instances, weights for each marker may be defined based on a reference dataset, or "training dataset".

[0053] These methods of analysis may be readily performed by one of ordinary skill in the art by employing a computer-based system, e.g. using any hardware, software and data storage medium as is known in the art, and employing any algorithms convenient for such analysis. For example, data mining algorithms can be applied through "cloud computing", smartphone based or client-server based platforms, and the like.

[0054] In certain embodiments the expression, e.g. polypeptide level, of only one marker is evaluated to produce a marker level representation. In yet other embodiments, the levels of two or more, i.e. a panel, markers, is evaluated. Accordingly, in the subject methods, the expression of at least one marker in a sample is evaluated. In certain embodiments, the evaluation that is made may be viewed as an evaluation of the proteome, as that term is employed in the art.

[0055] In some instances, the subject methods of determining or obtaining a preeclampsia marker representation (e.g. preeclampsia profile or preeclampsia score) for a subject further comprise providing the preeclampsia marker representation as a report. Thus, in some instances, the subject methods may further indude a step of generating or outputting a report providing the results of a preeclampsia marker evaluation in the sample, which report can be provided in the form of an electronic medium (e.g., an electronic display on a computer monitor), or in the form of a tangible medium (e.g., a report printed on paper or other tangible medium). Any form of report may be provided, e.g. as known in the art or as described in greater detail below.

UTILITY

[0056] Preeclampsia marker level representations so obtained find many uses. For example, the marker level representation may be employed to diagnose a preeclampsia; that is, to provide a determination as to whether a subject is affected by preeclampsia, the type of preeclampsia, the severity of preeclampsia, etc. In some instances, the subject may present with dinical symptoms of preeclampsia, e.g. elevated blood pressure (e.g. 140/90 mm/Hg or higher), proteinuria, sudden weight gain (over 1 -2 days or more than 2 pounds a week), water retention (edema), elevated liver enzymes, and/or thrombocytopenia (a depressed platelet count less than 100,000). In other instances, subject may be asymptomatic for preeclampsia but has risk factors associated with preeclampsia, e.g. a medical condition such as gestational diabetes, type I diabetes, obesity, chronic hypertension, renal disease, a thrombophilia; African-American or Filipino descent; age of greater than 35 years or less than 20 years; a family history of preeclampsia; nulliparity; preeclampsia in a previous pregnancy; and/or stress. In yet other instances, the subject may be asymptomatic for preeclampsia and have no risk factors associated with preeclampsia.

[0057] As another example, the preeclampsia marker level representation may be employed to prognose a preeclampsia; that is, to provide a preeclampsia prognosis. For example, the preeclampsia marker level representation may be used to predict a subject's susceptibility, or risk, of developing preeclampsia. By "predicting if the individual will develop preeclampsia", it is meant determining the likelihood that an individual will develop preeclampsia in the next week, in the next 2 weeks, in the next 3 weeks, in the next 5 weeks, in the next 2 months, in the next 3 months, or during the remainder of the pregnancy. The preeclampsia marker level representation may be used to predict the course of disease progression and/or disease outcome, e.g. expected onset of the preeclampsia, expected duration of the preeclampsia, expectations as to whether the preeclampsia will develop into edampsia, etc. The preeclampsia marker level representation may be used to predict a subject’s responsiveness to treatment for the preeclampsia, e.g., positive response, a negative response, no response at all.

[0058] As another example, the preeclampsia marker level representation may be employed to monitor a preeclampsia. By "monitoring" a preeclampsia, it is generally meant monitoring a subject’s condition, e.g. to inform a preeclampsia diagnosis, to inform a preeclampsia prognosis, to provide information as to the effect or efficacy of a preeclampsia treatment, and the like.

[0059] As another example, the preeclampsia marker level representation may be employed to determine a treatment for a subject. The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a mammal, and indudes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during or after the onset of disease or injury. The treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. The subject therapy may be administered prior to the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease. The terms "individual," "subject," "host," and "patient," are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. Preedampsia treatments are well known in the art, and may include bed rest, drinking extra water, a low salt diet, medicine to control blood pressure, corticosteroids, inducing pregnancy, and the like.

[0060] In some embodiments, the subject methods of providing a preeclampsia assessment, e.g. diagnosing a preeclampsia, prognosing a preeclampsia, monitoring the preeclampsia, treating the preeclampsia, and the like, may comprise comparing the obtained preeclampsia marker level representation to a preeclampsia phenotype determination element to identify similarities or differences with the phenotype determination element, where the similarities or differences that are identified are then employed to provide the preeclampsia assessment, e.g. diagnose the preeclampsia, prognose the preeclampsia, monitor the preeclampsia, determine a preeclampsia treatment, etc. By a "phenotype determination element" it is meant an element, e.g. a tissue sample, a marker profile, a value (e.g. score), a range of values, and the like that is representative of a phenotype (in this instance, a preeclampsia phenotype) and may be used to determine the phenotype of the subject, e.g. if the subject is healthy or is affeded by preeclampsia, if the subject has a preeclampsia that is likely to progress to eclampsia, if the subject has a preeclampsia that is responsive to therapy, etc.

[0061] For example, a preeclampsia phenotype determination element may be a sample from an individual that has or does not have preeclampsia, which may be used, for example, as a reference/control in the experimental determination of the marker level representation for a given subject. As another example, a preeclampsia phenotype determination element may be a marker level representation, e.g. marker profile or score, which is representative of a preeclampsia state and may be used as a reference/control to interpret the marker level representation of a given subject. The phenotype determination element may be a positive reference/control, e.g., a sample or marker level representation thereof from a pregnant woman that has preeclampsia, or that will develop preeclampsia, or that has preeclampsia that is manageable by known treatments, or that has preeclampsia that has been determined to be responsive only to the delivery of the baby. Alternatively, the phenotype determination element may be a negative reference/control, e.g. a sample or marker level representation thereof from a pregnant woman that has not developed preeclampsia, or an woman that is not pregnant. Phenotype determination elements are preferably the same type of sample or, if marker level representations, are obtained from the same type of sample as the sample that was employed to generate the marker level representation for the individual being monitored. For example, if the serum of an individual is being evaluated, the phenotype determination element would preferably be of serum.

[0062] In certain embodiments, the obtained marker level representation is compared to a single phenotype determination element to obtain information regarding the individual being tested for preeclampsia. In other embodiments, the obtained marker level representation is compared to two or more phenotype determination elements. For example, the obtained marker level representation may be compared to a negative reference and a positive reference to obtain confirmed information regarding if the individual will develop preeclampsia. As another example, the obtained marker level representation may be compared to a reference that is representative of a preeclampsia that is responsive to treatment and a reference that is representative of a preeclampsia that is not responsive to treatment to obtain information as to whether or not the patient will be responsive to treatment.

[0063] The comparison of the obtained marker level representation to the one or more phenotype determination elements may be performed using any convenient methodology, where a variety of methodologies are known to those of skill in the art. For example, those of skill in the art of ELISAs will know that ELISA data may be compared by, e.g. normalizing to standard curves, comparing normalized values, etc. The comparison step results in information regarding how similar or dissimilar the obtained marker level profile is to the control/reference profile(s), which simiiarity/dissimiiarity information is employed to, for example, predict the onset of a preeclampsia, diagnose preeclampsia, monitor a preeclampsia patient, etc. Similarly, those of skill in the art of arrays will know that array profiles may be compared by, e.g., comparing digital images of the expression profiles, by comparing databases of expression data, etc. Patents describing ways of comparing expression profiles indude, but are not limited to, U.S. Patent Nos. 6,308,170 and 6,228,575, the disclosures of which are herein incorporated by reference. Methods of comparing marker level profiles are also described above. Similarity may be based on relative marker levels, absolute marker levels or a combination of both. In certain embodiments, a similarity determination is made using a computer having a program stored thereon that is designed to receive input for a marker level representation obtained from a subject, e.g., from a user, determine similarity to one or more reference profiles or reference scores, and return an preeclampsia prognosis, e.g., to a user (e.g., lab technician, physician, pregnant individual, etc.). Further descriptions of computer-implemented aspects of the invention are described below. In certain embodiments, a similarity determination may be based on a visual comparison of the marker level representation, e.g. preeclampsia score, to a range of phenotype determination elements, e.g. a range of preeclampsia scores, to determine the reference preeclampsia score that is most similar to that of the subject. Depending on the type and nature of the phenotype determination element to which the obtained marker level profile is compared, the above comparison step yields a variety of different types of information regarding the cell/bodily fluid that is assayed. As such, the above comparison step can yield a positive/negative prediction of the onset of preeclampsia, a positive/negative diagnosis of preeclampsia, a characterization of a preeclampsia, information on the responsiveness of a preeclampsia to treatment, and the like.

[0064] In other embodiments, the marker level representation is employed directly, i.e. without comparison to a phenotype determination element, to make a preeclampsia prognosis, preeclampsia diagnosis, or monitor a preeclampsia.

[0065] The subject methods may be employed for a variety of different types of subjects. In many embodiments, the subjects are within the class mammalian, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), lagomorpha (e.g. rabbits) and primates (e.g., humans, chimpanzees, and monkeys). In certain embodiments, the animals or hosts, i.e., subjects (also referred to herein as patients), are humans.

[0066] In some embodiments, the subject methods of providing a preeclampsia assessment include providing a diagnosis, prognosis, or result of the monitoring. In some embodiments, the preeclampsia assessment of the present disclosure is provided by providing, i.e. generating, a written report that includes the artisan's assessment, for example, the artisan's determination of whether the patient is currently affected by preeclampsia, of the type, stage, or severity of the subjed's preeclampsia, etc. (a "preeclampsia diagnosis"); the artisan's prediction of the patient's susceptibility to developing preeclampsia, of the course of disease progression, of the patient’s responsiveness to treatment, etc. (i.e., the artisan’s "preeclampsia prognosis"); or the results of the artisan’s monitoring of the preeclampsia. Thus, the subject methods may further include a step of generating or outputting a report providing the results of an artisan's assessment, which report can be provided in the form of an electronic medium (e.g., an electtonic display on a computer monitor), or in the form of a tangible medium (e.g., a report printed on paper or other tangible medium). Any form of report may be provided, e.g. as known in the art or as described in greater detail below.

REPORTS

[0067] A "report," as described herein, is an electronic or tangible document which includes report elements that provide information of interest relating to the assessment of a subject and its results. In some embodiments, a subject report includes at least a preeclampsia marker representation, e.g. a preeclampsia profile or a preeclampsia score, as discussed in greater detail above. In some embodiments, a subject report includes at least an artisan's preeclampsia assessment, e.g. preeclampsia diagnosis, preeclampsia prognosis, an analysis of a preeclampsia monitoring, a treatment recommendation, etc. A subject report can be completely or partially electronically generated. A subject report can further include one or more of: 1 ) information regarding the testing facility; 2) service provider information; 3) patient data; 4) sample data; 5) an assessment report, which can include various information including: a) reference values employed, and b) test data, where test data can include, e.g., a protein level determination; 6) other features.

[0068] The report may include information about the testing facility, which information is relevant to the hospital, clinic, or laboratory in which sample gathering and/or data generation was conducted. Sample gathering can include obtaining a fluid sample, e.g. blood, saliva, urine etc.; a tissue sample, e.g. a tissue biopsy, etc. from a subject. Data generation can include measuring the marker concentration in preeclampsia patients versus healthy individuals, i.e. individuals that do not have and/or do not develop preeclampsia. This information can include one or more details relating to, for example, the name and location of the testing facility, the identity of the lab technician who conducted the assay and/or who entered the input data, the date and time the assay was conducted and/or analyzed, the location where the sample and/or result data is stored, the lot number of the reagents (e.g., kit, etc.) used in the assay, and the like. Report fields with this information can generally be populated using information provided by the user.

[0069] The report may include information about the service provider, which may be located outside the healthcare facility at which the user is located, or within the healthcare facility. Examples of such information can include the name and location of the service provider, the name of the reviewer, and where necessary or desired the name of the individual who conducted sample gathering and/or data generation. Report fields with this information can generally be populated using data entered by the user, which can be selected from among pre-scripted selections (e.g., using a drop-down menu). Other service provider information in the report can include contact information for technical information about the result and/or about the interpretive report.

[0070] The report may include a patient data section, including patient medical history (which can include, e.g., age, race, serotype, prior preeclampsia episodes, and any other characteristics of the pregnancy), as well as administrative patient data such as information to identify the patient (e.g., name, patient date of birth (DOB), gender, mailing and/or residence address, medical record number (MRN), room and/or bed number in a healthcare facility), insurance information, and the like), the name of the patient's physician or other health professional who ordered the monitoring assessment and, if different from the ordering physidan, the name of a staff physician who is responsible for the patient's care (e.g., primary care physician).

[0071] The report may include a sample data section, which may provide information about the biological sample analyzed in the monitoring assessment, such as the source of biological sample obtained from the patient (e.g. blood, saliva, or type of tissue, etc.), how the sample was handled (e.g. storage temperature, preparatory protocols) and the date and time collected. Report fields with this information can generally be populated using data entered by the user, some of which may be provided as pre-scripted selections (e.g., using a drop-down menu). The report may indude a results section.

[0072] The report may include an assessment report section, which may include information generated after processing of the data as described herein. The interpretive report can include a prediction of the likelihood that the subject will develop preeclampsia. The interpretive report can include a diagnosis of preeclampsia. The interpretive report can include a characterization of preeclampsia. The assessment portion of the report can optionally also include a recommendation(s). For example, where the results indicate that preeclampsia is likely, the recommendation can include a recommendation that diet be altered, blood pressure medicines administered, etc., as recommended in the art.

[0073] It will also be readily appreciated that the reports can include additional elements or modified elements. For example, where electronic, the report can contain hyperlinks which point to internal or external databases which provide more detailed information about selected elements of the report. For example, the patient data element of the report can include a hyperlink to an electronic patient record, or a site for accessing such a patient record, which patient record is maintained in a confidential database. This latter embodiment may be of interest in an in-hospital system or in-clinic setting. When in electronic format, the report is recorded on a suitable physical medium, such as a computer readable medium, e.g., in a computer memory, zip drive, CD, DVD, etc.

[0074] It will be readily appredated that the report can include all or some of the elements above, with the proviso that the report generally includes at least the elements sufficient to provide the analysis requested by the user (e.g. a calculated preeclampsia marker level representation; a prediction, diagnosis or characterization of preeclampsia).

REAGENTS, SYSTEMS AND KITS

[0075] Also provided are reagents, systems and kits thereof for practicing one or more of the above-described methods. The subject reagents, systems and kits thereof may vary greatly. Reagents of interest include reagents specifically designed for use in producing the above- described marker level representations of preeclampsia markers from a sample, for example, one or more detection elements, e.g. antibodies or peptides for the detection of protein, oligonucleotides for the detection of nucleic acids, etc. In some instances, the detection element comprises a reagent to detect the expression of a single preeclampsia marker, for example, the detection element may be a dipstick, a plate, an array, or cocktail that comprises one or more detection elements, e.g. one or more antibodies, one or more oligonucleotides, one or more sets of PCR primers, etc. which may be used to detect the expression of one or more preeclampsia marker simultaneously,

[0076] One type of reagent that is specifically tailored for generating marker level representations, e.g. preeclampsia marker level representations, is a collection of antibodies that bind specifically to the protein markers, e.g. in an ELISA format, in an xMAP™ microsphere format, on a proteomic array, in suspension for analysis by flow cytometry, by western blotting, by dot blotting, or by immunohistochemistry. Methods for using the same are well understood in the art. These antibodies can be provided in solution. Alternatively, they may be provided pre-bound to a solid matrix, for example, the wells of a multi-well dish or the surfaces of xMAP microspheres.

[0077] Another type of such reagent is an array of probe nucleic acids in which the genes of interest are represented. A variety of different array formats are known in the art, with a wide variety of different probe structures, substrate compositions and attachment technologies (e.g., dot blot arrays, microarrays, etc.). Representative array structures of interest include those described in U.S. Patent Nos.: 5,143,854; 5,288,644; 5,324,633;

5,432,049; 5,470,710; 5,492,806; 5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661,028; 5,800,992; the disclosures of which are herein incorporated by reference; as well as WO 95/21265; WO 96/31622; WO 97/10365; WO 97/27317; EP 373203; and EP 785 280.

[0078] Another type of reagent that is specifically tailored for generating marker level representations of genes, e.g. preeclampsia genes, is a collection of gene specific primers that is designed to selectively amplify such genes (e.g., using a PCR-based technique, e.g., real-time RT-PCR). Gene specific primers and methods for using the same are described in U.S. Patent No. 5,994,076, the disclosure of which is herein incorporated by reference.

[0079] Of particular interest are arrays of probes, collections of primers, or collections of antibodies that include probes, primers or antibodies (also called reagents) that are specific for at least 1 gene/protein/lipd selected from the group consisting of LEP, Ceramide (d 18: 1/25:0), Ceramide (d18: 1/26:0), or a biochemical substrate specific for the cofactor/prosthetic group heme. In certain embodiments, the collection of probes, primers, or antibodies includes reagents specific for LEP, Ceramide (d18:1/25:0), Ceramide (d 18: 1/26:0) as well as a biochemical substrate specific for heme. The subject probe, primer, or antibody collections or reagents may include reagents that are specific only for the genes/proteins/lipids/cofactors that are listed above, or they may include reagents specific for additional genes/proteins/lipids/cofactors that are not listed above, such as probes, primers, or antibodies specific for genes/proteins/lipids/cofactors whose expression pattern are known in the art to be associated with preeclampsia, e.g. and sFlt-1 (VEGF-RI) and PIGF.

[0080] In some instances, a system may be provided. As used herein, the term “system" refers to a collection of reagents, however compiled, e.g., by purchasing the collection of reagents from the same or different sources. In some instances, a kit may be provided. As used herein, the term "kit" refers to a collection of reagents provided, e.g., sold, together. For example, the nucleic acid- or antibody-based detection of the sample nucleic add or protein, respectively, may be coupled with an electrochemical biosensor platform that will allow multiplex determination of these biomarkers for personalized preeclampsia care.

[0081] The systems and kits of the subject invention may indude the above-described arrays, gene-specific primer collections, or protein-specific antibody collections. The systems and kits may further indude one or more additional reagents employed in the various methods, such as primers for generating target nudeic adds, dNTPs and/or rNTPs, which may be either premixed or separate, one or more uniquely labeled dNTPs and/or rNTPs, such as biotinylated or Cy3 or Cy5 tagged dNTPs, gold or silver partides with different scattering spectra, or other post synthesis labeling reagent, such as chemically active derivatives of fluorescent dyes, enzymes, such as reverse transcriptases, DNA polymerases, RNA polymerases, and the like, various buffer mediums, e.g. hybridization and washing buffers, prefabricated probe arrays, labeled probe purification reagents and components, like spin columns, etc., signal generation and detection reagents, e.g. labeled secondary antibodies, streptavidin-alkaline phosphatase conjugate, chemifluorescent or chemiluminescent substrate, and the like.

[0082] The subject systems and kits may also indude one or more preeclampsia phenotype determination elements, which element is, in many embodiments, a reference or control sample or marker representation that can be employed, e.g., by a suitable experimental or computing means, to make a preeclampsia prognosis based on an "input" marker level profile, e.g., that has been determined with the above described marker determination element. Representative preeclampsia phenotype determination elements include samples from an individual known to have or not have preeclampsia, databases of marker level representations, e.g., reference or control profiles or scores, and the like, as described above.

[0083] In addition to the above components, the subject kits will further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Yet another means would be a computer readable medium, e.g., diskette, CD, etc., on which the information has been recorded. Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.

[0084] The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES

[0085] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1

[0086] As the leading cause of maternal morbidity and mortality, preeclampsia (PE) is a pregnancy-related vascular disorder affecting 5%-8% of all pregnancies (Berg et al. Overview of maternal morbidity during hospitalization for labor and delivery in the United States: 1993-1997 and 2001-2005. Obstetrics and gynecology 2009;113:1075-81; Mackay et al. Pregnancy-related mortality from preeclampsia and eclampsia. Obstetrics and gynecology 2001 ;97:533-8). PE, which often causes fetal growth restriction and pre-term delivery as well as fetal mortality and morbidity, can be remedied by delivery of the placenta and fetus (Powe et al. Preeclampsia, a disease of the maternal endothelium: the role of antiangiogenic factors and implications for later cardiovascular disease. Circulation 2011;123:2856-69). The etiology of PE is incompletely understood. Current diagnosis of PE is based on the signs of hypertension and proteinuria (Gynecologists ACOOA ACOG practice bulletin. Diagnosis and management of preeclampsia and eclampsia. Number 33, January 2002. Obstetrics and gynecology 2002;99:159-67), but lacks sensitivity and specificity and carries a poor prognosis for adverse maternal and fetal outcomes (Zhang et al. Prediction of adverse outcomes by common definitions of hypertension in pregnancy. Obstetrics and gynecology 2001 ;97:261-7). Thus, there is a need to identify PE biomarkers that can provide a definitive diagnosis with the opportunity for better monitoring of the condition’s progression, and feus improved outcomes and economic benefits.

[0087] Although the pathophysiology remains largely elusive, PE is a multisystem disorder of pregnancy with the placenta playing a pivotal role. Investigators have used genetic, genomic, proteomic, and lipidomic approaches to compare PE and control placental tissues. Transcriptional profiling of case-control samples has identified disease-specific expression patterns, canonical pathways and gene-gene networks (Lapaire et al. Microarray screening for novel preeclampsia biomarker candidates. Fetal diagnosis and therapy 2012;31:147-53; Nishizawa et al. Microarray analysis of differentially expressed fetal genes in placenta tissue derived from early and late onset severe preeclampsia. Placenta 2007;28:487-97; Loset et al. transcriptional profile of the decidua in preeclampsia. American journal of obstetrics and gynecology 2011 ;204:84 e1-27; Johansson et al. Partial correlation network analyses to detect altered gene interactions in human disease: using preeclampsia as a model. Human genetics 2011;129:25-34; Sitras et al. Differential placental gene expression in severe preeclampsia. Placenta 2009;30:424-33; Tsai et al. Transcriptional profiling of human placentas from pregnancies complicated by preeclampsia reveals disregulation of sialic acid acetylesterase and immune signaling pathways. Placenta 2011;32:175-82; Winn et al. Severe preeclampsia-related changes in gene expression at fee maternal-fetal interlace include sialic acid-binding immunoglobulin-like lectin-6 and pappalysin-2. Endocrinology 2009;150:452-62). Preeclampsia-related biomarker studies (Kolia et al. Quantitative proteomic (iTRAQ) analysis of 1st trimester maternal plasma samples in pregnancies at risk for preeclampsia. Journal of biomedicine & biotechnology 2012:2012:305964; Mary et al. Dynamic proteome in enigmatic preeclampsia: an account of molecular mechanisms and biomarker discovery. Proteomics Clinical applications 2012;6:79-90; Carty et al. Urinary proteomics for prediction of preeclampsia. Hypertension 2011 ;57:561-9; Ho et al. ELABELA deficiency promotes preeclampsia and cardiovascular malformations in mice. Sdence 2017) have also revealed candidate biomarkers for future testing. Placental angiogenic and anti-angiogenic factor imbalance, elevated soluble fms-like tyrosine kinase (sFlt-1 ) and decreased placental growth factor (PIGF) levels, are suggested in the pathogenesis of PE (Shibata et al. Soluble fms-like tyrosine kinase 1 is increased in preeclampsia but not in normotensive pregnancies with small-for-gestational-age neonates: relationship to circulating placental growth factor. The Journal of clinical endocrinology and metabolism 2005;90:4895-903; Maynard et al. Excess placental soluble fms-like tyrosine kinase 1 (sFlt-1 ) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. The Journal of clinical investigation 2003;111:649-58; Wolf et al. Circulating levels of the antiangiogenic marker sFLT-1 are increased in first versus second pregnancies. American journal of obstetrics and gynecology 2005;193:16-22; Rajakumar et al. Extra-placental expression of vascular endothelial growth factor receptor- 1 , (Flt-1 ) and soluble Flt-1 (sFlt-1), by peripheral blood mononuclear cells (PBMCs) in normotensive and preeclamptic pregnant women. Placenta 2005;26:563-73; Taylor et al. Altered tumor vessel maturation and proliferation in placenta growth factor- producing tumors: potential relationship to post-therapy tumor angiogenesis and recurrence. International journal of cancer Journal international du cancer 2003;105:158-64; Tidewell et al. Low maternal serum levels of placenta growth factor as an antecedent of clinical preeclampsia. American journal of obstetrics and gynecology 2001 ;184:1267-72; Tony et al. Preeclampsia is associated with reduced serum levels of placenta growth factor. American journal of obstetrics and gynecology 1998;179:1539-44), and the sFlt-1/PIGF ratio has been proposed as a useful index in the diagnosis and management of PE (Stepan et al. [use of angiogenic factors (sflt-1 Zplgf ratio) to confirm the diagnosis of preeclampsia in clinical routine: First experience]. Zeitschrift fur Geburtshilfe und Neonatologie. 2010;214:234-238; Veriohren et al. An automated method for the determination of the sflt-1/PIGF ratio in the assessment of preeclampsia. Am. J. Obst. And Gyn. 2010;202:161 e161-161 e111). However, no widely applicable, sensitive and specific molecular PE test in routine clinical practice is currently available.

[0088] In light of these considerations, there is a strong rationale and need to discover diagnostic and prognostic biomarkers for PE. We employed a comprehensive unbiased multi-'omics’ approach, integrating results from microarray multiplex meta-analysis, and proteomic identification by two-dimensional (2D) gel analysis as well as mass spectrometry detection and quantification. Our applied parametric method (Morgan et al. Comparison of multiplex meta analysis techniques for understanding the acute rejection of solid organ transplants. BMC bioinformatics 2010;11 Suppl 9:S6; Chen etal. Differentially expressed RNA from public microarray data identifies serum protein biomarkers for cross-organ transplant rejection and other conditions. PLoS computational biology 2010;6) in meta-analysis allowed us to identify consistent and significant differential gene expression across experiments to develop biomarkers for downstream experimental validation. Serum proteins are routinely used to diagnose diseases, but sensitive and specific biomarkers are hard to find and may be due to their low serological abundance, which can easily be masked by highly abundant proteins. Our serum protein marker discovery method (Ling et al. Plasma profiles in active systemic juvenile idiopathic arthritis: Biomarkers and biological implications. Proteomics 2010) combines antibody-based serum abundant protein depletion and 2D gel comparative profiling to discover differential protein gel spots between PE and control sera for subsequent protein mass spectrometric identification. We hypothesized that there would be differential serological signatures allowing PE diagnosis. To construct and optimize a sensitive and specific biomarker panel with the least number of protein analytes, a binary classification algorithm was used. Close examination of the biomarkers from comparative transcriptomics and proteomics, and their associated pathways led to new hypothesis about their role in PE pathophysiology. We filed a USPTO patent application on assessment of PE using Leptin itself (Application number: 62757099; 2018).

[0089] The presented results validated our hypothesis that sensitive and specific serological biomarker panels can be constructed to diagnose PE. To our knowledge, this represents the first study to uncover novel PE biomarkers including LEP and ceramides in PE discrimination. We believe that the functional significance of these PE biomarkers will provide new insights into the disease pathogenesis and lead to effective novel therapeutics.

MATERIALS AND METHODS

[0090] Study design. The overall sample allocation, PE biomarker discovery, validation, and predictive panel construction steps are illustrated in Figure 1. Our study was conducted in two phases: (1) the discovery phase (Hypothesis generation). (2) the validation phase.

[0091] Hypothesis generation. Our previously serologic tests on placental-related proteins demonstrated that LEP is a potential marker to assess PE in early stage of gestation. A literature review was conducted in parallel to identify markers associated with PE, especially on ceramides which are downstream of LEP. The results of the two approaches were integrated to generate a hypothesis that LEP and ceramides can be used for PE assessment.

[0092] Measurement of PE marker candidates using ELISA. ELISA was performed on samples using commercial kits following vendors' instructions. Assays were performed to measure serum level of LEP, sFlt-1, and PIFG. Two types of ELISA were applied, sandwich ELISA and competitive ELISA. Briefly, for sandwich ELISA, the capture antibody has been pre-coated onto the microplate and then standards and serum samples are added into the wells of the microplate to bind with the capture antibody. After extensive washing to avoid nonspecific binders, a second detection antibody conjugated with horseradish peroxidase (HRP) was added to the wells. After extensive washing to remove any unbound antibody-enzyme reagent, a HRP substrate solution followed by stop solution is added to the microplate wells. The optical density (O.D.) of the microplate wells is measured and the O.D. is proportional to the amount of analyte present in the sample. The sample analyte concentration is calculated based on standard curve. For competitive ELISA, the capture antibody for antiserum is pre-coated onto the microplate. A constant concentration of biotinylated tracer (Bt-tracer) and varying concentrations of unlabeled standard or sample peptide are added into the wells and they compete for binding specifically to the antiserum. After extensive washing to avoid nonspecific binder and remove unbound reagents, streptavidin-conjugated HRP is added into the wells to bind Bt-tracer specifically, which produces a soluble colored product after a substrate is added. The optical density (O.D.) of the microplate wells is measured and the O.D. is inverse proportional to the amount of analyte present in the sample.

[0093] Measurement of PE marker candidates using mass spectrometry. A high-throughput UPLC/MS/MS method was developed to quantify serum level of Ceramides.

[0094] Step 1. Materials. The calibration standard ceramide (d18: 1/24:0) and stable isotope labeled internal standards d7-ceramide (d18:1/24:0) were purchased from Avanti Lipids (Alabaster, AL). HPLC grade water, methanol, 2-propanol, and chloroform were obtained from Fisher Scientific (Pittsburgh, PA). Analytical grade ammonium bicarbonate was purchased from Sigma Aldrich (St. Louis, MO). The de-lipidized serum VD-DDC Mass Spec Gold was obtained from Golden West Biological (Temecula, CA). All materials were directly used without further purification.

[0095] Step 2. MRM Transition Optimization. The MRM transitions for targeted ceramides and dihydroceramides were individually optimized by direct syringe pump infusion of 0.50 uM of the corresponding standard at 10 μL/min into the mass spectrometer in the presence of 10 mM of ammonium bicarbonate. The SRM transitions were optimized and recorded for parent ion m/z, daughter ion m/z, collision energy, and RF lens on a Thermo TSQ Quantiva mass spectrometer. The Q1 and Q3 resolutions were both set at 0.7 Da.

[0096] Step 3. Sample Preparation. For the preparation of blank, 10-μL aliquot of de-lipidized serum was spiked with 10 μL of 2-propanol to obtain blank sample. The blank samples were extracted with 200 μL of methanol and internal standard working solution to obtain double and single blanks, respectively. For the preparation of calibrator, 10- μL aliquot of de-lipidized serum was spiked with 10 μL of calibrator working solution to obtain the calibrator at the corresponding level. The spiked calibrators were individually extracted with 200 μL of internal standard working solution to obtain a set of calibrators based on 6 concentration levels. For the preparation of QC, 10-μL aliquot of de-lipidized serum was spiked with 10 μL of QC working solutions to obtain the QC at the corresponding level. The spiked QCs were individually extracted with 200 of internal standard working solution to obtain a set of QCs based on 4 concentration levels. For the preparation of serum sample, 10- μL aliquot of unknown sample was spiked with 10 μL of 2-propanol and extracted with 200 μL of internal standard working solution. Following the extraction, all extracted samples were subject to vigorous vortex for 30 secs and high-speed centrifuge at 12,000xg under 4 °C for 5 mins. Thereafter, 180 μL of supernatant was removed from each sample and transferred into an auto-sampler vial with micro-insert for LC/MS analysis.

[0097] Step 4. LC-MS analysis. Following sample preparation, 10 μL of the sample was injected onto an Thermo Ultimate 3000 UPLC system equipped with an ACE Excel SuperC18 column (1.7 μm, 100 mmx2.1 mm; MAC-MOD Analytical, Chadds, PA). The mobile was composed of a mixture of methanol and 2-propanol at 1 :1 buffered by 10 mM of ammonium bicarbonate. Chromatographic separation was carried out using a 5-min isocratic elusion program. Briefly, the LC eluent was directed to the waste for the first 1.0 min and then switched back to the electrospray interface from 1.1 to 5 min, allowing the targeted ceramides and dihydroceramides to be sequentially eluted, ionized, and detected by the system. The flow rate was set constantly at 0.3 mL/min, and the temperatures of auto-sampler and column oven were maintained at 4 and 30 °C, respectively, throughout the analysis. The mass spectrometer was operated in a scheduled multiple reaction monitoring (MRM) mode to continuously acquire data from the LC eluent. The retention time-dependent data acquisition was employed using pre-defined retention time windows with variable widths (1.2 mins for medium drain and 1.5 mins for long chain ceramides and dihydroceramides) to record the extracted ion chromatograms (EIC) of targeted analytes.

[0098] Setp 5. Quantitation. Chromatographic peak integration and analyte quantitation were implemented using the XCalibur 4.0 software package from Thermo Fisher. The IS-normalized peak area ratios from the quantitative ions were plotted against the spiked concentrations in calibrators to establish calibration curves based on 6 levels. The linear regression fitting with a weighting factor of 1/x² was employed for the calibration. Thereafter, a >=0.99 cutoff was set on the square of correlation coefficient to ensure the calibration curve is qualified for quantitation. Afterwards, the IS-normalized peak area ratios of targeted analytes were plugged into the corresponding calibration curves to obtain absolutely quantitated concentrations in blank, QC, and serum samples.

[0099] Statistical analyses. Patient demographic and clinical data were analyzed using the "Epidemiological calculator" (R epicalc package). Student’s t-test and Mann- Whitney U-test were performed to calculate p values for continuous variables, and Fisher’s exact test and Chi-squared test were used for comparative analysis of categorical variables. A group of clinical risk factors of preeclampsia were determined by literature review, and their impacts on preeclampsia diagnosis were explored by uni- and multivariate analysis. Serum protein statistical analysis provides an overall effect estimation of each analyte's capability in discriminating PE and normal pregnant control subjects. Hypothesis testing was performed using Student's t-test (two tailed) and Mann-Whitney U-test (two tailed), and local FDR (Efron et al. Empirical bayes analysis of microarray experiment. J Am Stat Assoc 2001 ;96:1151 -60) to correct for multiple hypothesis testing issues. The predictive performance of each biomarker panel analysis was evaluated by ROC curve analysis (Zweig et al. Receiveroperating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine. Clinical chemistry 1993;39:561-77; Sing et al. ROCR: visualizing classifier performance in R. Bioinformatics 2005;21:3940-1). The biomarker panel score was defined as the natural logarithm of the ratio between the geometric means of the respective up- and down-regulated protein biomarkers in the maternal circulation. A composite panel combining LEP, APO-A1 , FGA, pikachurin, and PIFG was derived as PE classification panel where the score was calculated as the ratio of LEP and Ceramide (d18:1/25:0), or the ratio of LEP and Ceramide (d 18: 1/26:0), and evaluated by ROC AUG performance. The ratio of sFItl and PIGF was used as a reference.

RESULTS

[00100] Study design of exploring and validating PE marker candidates. As shown in Figure 1 , LEP, ceramides, and the ratios of LEP to ceramides were measured on a discovery cohort (PE=32, control=32) where serum samples of PE patients were collected after confirmative diagnosis, and the binary classification performance of the markers and the ratios were explored. The levels of LEP, ceramides, and the ratios of LEP to ceramides in serum were then tested on an independent, longitudinal cohort (PE=20, control=20), and their performance of assessing the PE prior to confirmative diagnosis of PE was evaluated. The ratio of sFItl and PIGF was used as a reference biomarker.

[00101] Sample characteristics. The baseline characteristics of PE and control subjects were shown in Tables 1 and 2. In the discovery cohort, each patient had one sample collected between 24-40 wks of gestation. In the testing cohort, each patient had 1-3 samples collected at the 1^st, 2^nd, and 3^rd trimesters (Figure 2).

Table 1. Patient characteristics of the discovery cohort. Early stage: Patients having blood draw at 24-33 wks. Late stage: Patients having blood draw at 34-40 wks.

•The time of blood drawing. PE, preecfamptia.

Table 2. Patient characteristics of the testing cohort.

[00102] Evaluating biomarkers for PE assessment on the discovery cohort

Levels of LEP 16 ceramides, 10 dihydroceramides were measured in each sample. A significant up-regulation of LEP was observed at 24-28 wks of gestation in PE patients

(P=0.02; fold of change=3.3; Figure 3). Ceramide (d18:1/25:0) and (d18:1/26:0) were found slightly down-regulated at 24-28 wks (fold of changes=0.8; Figure 3). The LEP/Ceramide

(d 18: 1/25:0) ratio and LEP/Ceramide (d18:1/26:0) ratio were both significantly upregulated in

PE patients at 24-33 wks of gestation (P=0.02 for both ratios; Figure 4). The AUG of the ratios are both 0.83 at 24-33 wks. [00103] Evaluating biomarkers for impending PE assessment on the testing cohort. To identify whether the PE serological protein panel could enable development of an immediate practical clinical tool, LEP and two ceramides were tested using a longitudinal cohort where samples were collected between 5-29 wks and prior to confirmative diagnosis of PE. Significant upregulations of LEP were observed at 10-14 wks (P=0.02) and 15-25 wks (P=3x10^-6) of PE patients (Figure 5). Ceramide d18:1/25:0 and d18:1/26:0 were down-regulated in PE patients over 5-29 wks, with significant differences between PE patients and controls were observed at 16-20 wks (P=0.004 for d18: 1/25:0 and P=0.0009 for d18:1/26:0) and 21-29 wks (P=0.002 for d18:1/25:0 and P=0.0009 for d18:1/26:0). The BMI-Normalized LEP/Ceramide (d18:1/25:0) ratio showed significant differences between PE patients and controls over 5-29 wks of gestation: P=0.005 at 5-11 wks; P=5x10^-4 at 12-15 wks and 16-20 wks; P=6x10^-4 at 21-29 wks. The BMI-Normalized LEP/Ceramide (d 18:1/26:0) ratio had similar trends: P=0.02 at 5-11 wks; P=0.001 at 12-15 wks; P=7x10^-4 at 16-20 wks; P=4x10^-4 at 21-29 wks (Figure 6). The AUG of the BMI-Normalized LEP/Ceramide ratio reached a peak at 12-15 wks (0.95 for d18:1/25:0 and 0.93 for d18:1/26:0; Figure 6). Compared with the reference point of the sFlt-1/PIGF ratio, the AUG of the BMI-Normalized LEP/Ceramide ratios were significantly better (P=0.002 for LEP/Ceramide (d18:1/25:0); P=0.001 for LEP/Ceramide (d18:1/26:0)).

[00104] Time-event analysis on the testing cohort. There were 17 of 20 patients had confirmative diagnostic dates of PE. Among the 17 patients, 9 (52.9%) were identified by the BMI-Normalized LEP/Ceramide (d18:1/25:0) ratio >23 weeks prior to the confirmative diagnosis of PE, and 16 (94.1%) were identified >15 weeks prior to the confirmative diagnosis of PE (Figure 7). The mean±SD of the gap between the identification and diagnosis was 23.9±4.0 weeks. Compared to the reference point sFlt-1/PIGF that only identified 4 patients at >15 weeks prior to the confirmative diagnosis, the LEP/Ceramide (d18:1/25:0) ratio was able to predict PE much earlier (P=0.01 ).

[00105] Sensitivity, specificity, PPV, and NPV on the testing cohort. The performance of impending PE assessment of BMI-normalized LEP/Ceramide (d 18: 1/25:0) and BMI-normalized LEP/Ceramide (d18:1/26:0) was measured by sensitivity, specificity, PPV, and NPV at 5-29 wks and 12-20 wks, respectively (Figure 8). BMI-normalized LEP/Ceramide (d18:1/25:0) had a sensitivity of 0.83, a specificity of 0.81 , a PPV of 0.80, and a NPV of 0.84 at 5-29 wks of GA. The performance of the marker was even better at 12-20 wks when the AUG peaked: it had a sensitivity of 0.85, a specificity of 0.95, a PPV of 0.94, and a NPV of 0.86.

Claims

1. A method of providing a preeclampsia marker level representation for a subject, the method comprising: evaluating a panel of preeclampsia markers in a blood sample from a subject to determine the level of each preeclampsia marker in the blood sample; and obtaining the preeclampsia marker level representation based on the level of each preeclampsia marker in the panel, wherein the panel of preeclampsia markers comprise Leptin (LEP).

2. The method according to claim 1 , wherein the panel of preeclampsia markers further comprise one or more preeclampsia markers seleded from the group consisting of Ceamide

(d18:1/25:0), and Ceamide (d18:1/26:0).

3. The method according to claim 2, wherein the panel of preeclampsia markers comprises

Leptin (LEP) and Ceamide (d18: 1/25:0).

4. The method according to claim 2, wherein the panel of preeclampsia markers consists of

Leptin (LEP) and Ceamide (d18:1/26:0).

5. The method according to claim 1 , further comprising providing a report of the preeclampsia marker level representation.

6. The method according to claim 1 , wherein the preeclampsia marker representation is a preeclampsia score.

7. The method according to daim 2, wherein the preeclampsia marker representation is a ratio of LEP/ Ceamide (d18:1/25:0).

8. The method according to claim 2, wherein the preeclampsia marker representation is a ratio of LEP/ Ceamide (d18:1/26:0).

9. A method for providing a preeclampsia diagnosis for a subject, the method comprising: obtaining a preeclampsia marker level representation for a sample from a subject, and providing a preeclampsia diagnosis for the subject based on the preeclampsia marker level representation, wherein the preeclampsia marker level representation is obtained based on a level of each preeclampsia marker in a panel of preeclampsia markers comprising Leptin (LEP).

10. The method according to claim 9, wherein the preeclampsia marker level representation is based on the level of preeclampsia markers in the panel of preeclampsia markers further comprising one or more markers selected from the group consisting of Ceamide (d18:1/25:0), and Ceamide (d18:1/26:0).

11. The method according to claim 9, wherein the panel of preeclampsia markers comprises

LEP and Ceamide (d18:1/25:0).

12. The method according to claim 9, wherein the panel of preeclampsia markers comprises

LEP and Ceamide (d18:1/26:0).

13. The method according to claim 9, wherein preeclampsia marker level representation is a ratio of LEP/Ceamide (d18:1/25:0).

14. The method according to claim 9, wherein preeclampsia marker level representation is a ratio of LEP/Ceamide (d18:1/26:0).

15. The method according to claim 9, wherein the subject has symptoms of preeclampsia.

16. The method according to daim 9, wherein the subject is asymptomatic for preeclampsia.

17. The method according to claim 9, wherein the subject has risk factors associated with preeclampsia.

18. The method according to claim 9, wherein the preeclampsia is eariy-onset (<34 weeks of gestation).

19. The method according to claim 9, wherein the preeclampsia is late-onset (£34 weeks of gestation).

20. The method according to claim 9, wherein the sample is collected at 5-29 weeks of gestation.

21. The method according to claim 9, wherein the method further comprises comparing the preeclampsia marker level representation to a preeclampsia phenotype determination element, and providing a preeclampsia diagnosis for the subject based on the comparison.

22. The method of any one of daims 1-21 , wherein the subject:

(a) has history of preeclampsia;

(b) is older than 40;

(c) has babies less than two years or more than 10 years apart;

(d) has obesity; or

(e) has history of certain conditions including chronic high blood pressure, migraine headaches, type 1 or type 2 diabetes, kidney disease, a tendency to develop blood dots, or lupus.

23. The method of any one of daims 1-21 , further comprising administering to the subject identified as preeclampsia a procedure to ameliorate the preeclampsia.

24. The method of daim 23, wherein the procedure is selected from the group consisting of medications to lower blood pressure, use of corticosteroids, anticonvulsant medication such as magnesium sulfate, bed rest, and consideration of delivery if the diagnosis was made at or after 37 gestational weeks.

25. A method of treating a pregnant woman as at preeclampsia, comprising:

(a) measuring with antibodies, in a serum sample obtained from the woman, the expression levels of markers in a panel of preeclampsia markers comprising Leptin (LEP); and

(b) administering to the woman a procedure to ameliorate the preeclampsia when the woman is identified as preeclampsia.

26. The method of claim 25, wherein the panel of preeclampsia markers further comprises one or more markers selected from the group comprising Ceamide (d18: 1/25:0) and

Ceamide (d18:1/26:0).

27. The method of daim 25, wherein the procedure is selected from the group consisting of medications to lower blood pressure, use of corticosteroids, anticonvulsant medication such as magnesium sulfate, bed rest, and consideration of delivery if the diagnosis was made at or after 37 gestational weeks.

28. The method of daim 25, wherein the sample is collected at 5-29 weeks of gestation.

29. The method of claim 25, wherein the preeclampsia is eariy-onset or late-onset.

30. The method of claim 25, wherien the measurement is performed only for LEP, Ceamide

(d 18: 1/25:0), and Ceamide (d 18:1/26:0).

31. The method of claim 25, wherien the measurement is performed only for LEP and

Ceamide (d18: 1/25:0).

32. The method of claim 25, wherien the measurement is performed only for LEP and

Ceamide (d18:1/26:0).

33. The method of claim 25, wherien the measurement is performed only for Ceamide

(d 18: 1/25:0).

34. The method of claim 25, wherien the measurement is performed only for Ceamide

(d 18: 1/26:0).

35. The method of claim 25, wherien the measurement is performed only for LEP.

36. A kit for making a preeclampsia diagnosis, comprising:

(a) one or more detection elements for measuring the amount of marker in a sample for a panel of preeclampsia markers comprising Leptin (LEP).

37. The kit of claim 36, further comprising (b) a preeclampsia phenotype determination element.

38. The kit of claim 36, wherein the panel of preeclampsia markers further comprises one or more markers selected from the group consisting of LEP, Ceamide (d18:1/25:0), and

Ceamide (d18:1/26:0).

39. The kit of claim 36, wherein the one or more detection elements are antibodies directed to any one or more of the markers, probe nucleic adds directed to genes encoding any one or more of the markers, or gene specific primers directed to a fragment of a gene encoding any one or more of the markers.

40. The kit of claim 39, wherein the one or more detection elements comprising antibodies directed to no more than seven markers beside a control antibody.

41. The kit according to claim 36, wherein the panel of preeclampsia markers comprises LEP,

Ceamide (d18:1/25:0), and Ceamide (d18:1/26:0).

42. The kit according to claim 36, wherein the panel of preeclampsia markers comprises of LEP and Ceamide (d18:1/25:0).

43. The kit according to claim 36, wherein the panel of preeclampsia markers comprises of

LEP and Ceamide (d18:1/26:0).

44. The kit according to claim 36, wherein the panel of preeclampsia markers comprises of

Ceamide (d18:1/25:0).

45. The kit according to daim 36, wherein the panel of preeclampsia markers comprises of

Ceamide (d18:1/26:0).

46. The kit according to claim 36, wherein the panel of preeclampsia markers consists of

LEP.