Preeclampsia biomarkers and related systems and methods

Abstract

Disclosed herein are methods, kits, tests, and systems for detecting, predicting, monitoring, or ruling out preeclampsia in pregnant women. Also provided herein are novel diagnostic markers, methods of data analysis, assay formats, and kits employing such markers to improve one or more characteristics of a test for identifying or ruling out preeclampsia based on biomarkers from patient samples.

What is claimed is: 
     
         1 . A method for avoiding unnecessary treatment of preeclampsia, the method comprising:
 (a) contacting a biological sample that has been collected from a pregnant human female with a plurality of different probes, wherein the plurality of different probes comprises probes with specific affinity for four or more proteins selected from proteins listed in Table A or Table B;   (b) determining, based on binding of the plurality of different probes to corresponding proteins, an amount or concentration for each of the four or more proteins; and   (c) proceeding with treatment of said pregnant human in a manner that avoids unnecessary treatment of preeclampsia based at least in part on the amounts or concentrations of the four or more proteins determined in step (b).   
     
     
         2 . The method of  claim 1 , wherein the four or more proteins comprise:
 (a) placental growth factor (PlGF);   (b) one or more angiogenesis-associated proteins selected from the group consisting of soluble fms-like tyrosine kinase 1 (sFlt1), endoglin, pappalysin 2 (PAPP-A2), and decorin; and   (c) one or more kidney damage associated-proteins selected from the group consisting of (1) kidney injury molecule-1 (KIM1), (2) programmed cell death 1 ligand 1 (CD274), and (3) decorin.   
     
     
         3 . The method of  claim 2 , wherein the four or more proteins further comprise one or more proteins selected from the group consisting of C-type lectin domain family 4 member A (CLEC4A), fibroblast growth factor 21 (FGF21), trefoil factor 2 (TFF2), and hepatocyte growth factor (HGF). 
     
     
         4 . The method of any one of  claims 1 - 3 , wherein the four or more proteins comprise PlGF, sFlt1, KIM1, and CLEC4A. 
     
     
         5 . The method of any one of  claims 1 - 4 , wherein the plurality of different probes comprises probes with specific affinity to fibroblast growth factor 21 (FGF21), and the four or more proteins comprise FGF21. 
     
     
         6 . The method of any one of  claims 1 - 5 , wherein the plurality of different probes comprises probes with specific affinity for endoglin, and the four or more proteins comprise endoglin. 
     
     
         7 . The method of any one of  claims 1 - 6 , wherein the plurality of different probes comprises probes with specific affinity for decorin, and the four or more proteins comprise decorin. 
     
     
         8 . The method of any one of  claims 1 - 7 , wherein the plurality of different probes comprises probes with specific affinity for cluster of differentiation 274 (CD274), and the four or more proteins comprise CD274. 
     
     
         9 . The method of any one of  claims 1 - 8 , wherein the plurality of different probes comprises probes with specific affinity for hepatocyte growth factor (HGF), and the four or more proteins comprise HGF. 
     
     
         10 . The method of any one of  claims 1 - 9 , wherein the plurality of different probes comprises probes with specific affinity for trefoil factor 2 (TFF2), and the four or more proteins comprises TFF2. 
     
     
         11 . The method of any one of  claims 1 - 10 , wherein the plurality of different probes comprises probes with specific affinity for pappalysin-2 (PAPP-A2), and the four or more proteins comprises PAPP-A2. 
     
     
         12 . The method of any one of  claims 1 - 11 , wherein the biological sample is obtained from the pregnant female after gestational week 20. 
     
     
         13 . The method of any one of  claims 1 - 12 , wherein the biological sample has been collected from the pregnant female prior to gestational week 30. 
     
     
         14 . The method of any one of  claims 1 - 13 , further comprising:
 applying a classifier algorithm to an expression profile of the four or more proteins, wherein the classifier algorithm calculates an index; and   comparing the index to a reference value to determine whether to avoid the unnecessary treatment of preeclampsia.   
     
     
         15 . The method of any one of  claims 1 - 14 , wherein the classifier algorithm further comprises a correction for gestational age. 
     
     
         16 . The method of  claim 15 , wherein the correction for gestational age comprises a LOESS correction. 
     
     
         17 . The method of any one of  claims 14 - 16 , wherein the classifier algorithm comprises a logistic regression. 
     
     
         18 . The method of any one of  claims 14 - 17 , wherein the classifier algorithm comprises a logistic regression with elastic-net regularization. 
     
     
         19 . The method of any one of  claims 14 - 16 , wherein the classifier algorithm comprises a Random Forest. 
     
     
         20 . The method of any one of  claims 1 - 19 , wherein the biological sample is a urine, blood, amniotic fluid, exosome, plasma, or serum sample. 
     
     
         21 . The method of any one of  claims 1 - 19 , wherein the biological sample is from blood of the pregnant human female. 
     
     
         22 . The method of any one of  claims 1 - 21 , wherein the amount or concentration of no more than 20, no more than 15, nor more than 10, nor more than 9, no more than 8, no more than 7, no more than 6, nor more than 5, or no more than 4 proteins is determined. 
     
     
         23 . The method of any one of  claims 1 - 22 , wherein one or more of the plurality of different probes are antibodies or antibody fragments. 
     
     
         24 . The method of any one of  claims 1 - 23 , wherein each of the plurality of different probes are antibodies or antibody fragments. 
     
     
         25 . The method of any one of  claims 1 - 24 , wherein the amount or concentration of at least one of the four or more proteins is determined using a luminescent oxygen channeling immunoassay. 
     
     
         26 . The method of any one of  claims 1 - 25 , wherein the amount or concentration of at least one of the four or more proteins is determined using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. 
     
     
         27 . The method of any one of  claims 1 - 26 , wherein the amount or concentration of at least one of the four or more proteins is determined using a proximity extension assay. 
     
     
         28 . The method of any one of  claims 1 - 27 , wherein the amount or concentration of at least one of the four or more proteins is determined using an enzyme-linked immunosorbent assay (ELISA). 
     
     
         29 . The method of any one of  claims 1 - 28 , wherein the amount or concentration of at least one of the four or more proteins is determined using an amplified luminescent proximity homogenous assay. 
     
     
         30 . The method of any one of  claims 1 - 29 , wherein the amount or concentration of at least one of the four or more proteins is determined using a lateral flow assay. 
     
     
         31 . The method of any one of  claims 1 - 30 , wherein the biological sample is obtained from the pregnant human while in a perinatologist's office, a labor and delivery room, or triage (ER). 
     
     
         32 . The method of any one of  claims 1 - 31 , further comprising separating the biological sample into a plurality of different reaction vessels, the plurality of reaction vessels comprising a first reaction vessel, a second reaction vessel, a third reaction vessel, and a fourth reaction vessel, wherein contacting the biological sample with the plurality of different probes comprises delivering probes with specific affinity for PlGF in a first reaction vessel, delivering probes with specific affinity to sFlt1 to a second reaction vessel, delivering probes with specific affinity to KIM1 to a third reaction vessel, and delivering probes with specific affinity to CLEC4A to a fourth reaction vessel. 
     
     
         33 . The method of any one of  claims 1 - 29 , wherein the step of contacting the biological sample with the plurality of different probes occurs in a single reaction vessel. 
     
     
         34 . The method of any one of  claims 1 - 33 , wherein the biological sample was obtained from the pregnant female after the pregnant female has shown one or more symptoms of preeclampsia, wherein the symptoms of preeclampsia are selected from (1) high blood pressure and (2) proteinuria. 
     
     
         35 . The method of  claim 34 , wherein the sample was obtained from the pregnant female after the pregnant female has shown both (1) high blood pressure and (2) proteinuria. 
     
     
         36 . The method of any one of  claims 1 - 35 , wherein the plurality of different probes comprises two sets of probes with specific affinity for each of the four or more proteins, wherein each set of the two sets of probes binds to different epitopes. 
     
     
         37 . A method, such as a laboratory method, for detecting and/or quantifying a plurality of proteins in a sample from a pregnant human female, the method comprising:
 contacting a biological sample from a pregnant human female with a plurality of probes, wherein the plurality of probes comprises probes with specific affinity for four or more proteins selected from the proteins listed in Table A or Table B;   detecting the presence and/or quantity of the four or more proteins based on binding of the plurality of different probes to corresponding proteins.   
     
     
         38 . The method of  claim 37 , wherein the four or more proteins comprise:
 (a) placental growth factor;   (b) one or more angiogenesis-associated proteins selected from the group consisting of soluble fms-like tyrosine kinase 1 (sFlt1), endoglin, pappalysin 2 (PAPP-A2), and decorin; and   (c) one or more kidney damage associated-proteins selected from the group consisting of (1) kidney injury molecule-1 (KIM1), (2) programmed cell death 1 ligand 1 (CD274), and decorin.   
     
     
         39 . The method of  claim 38 , wherein the four or more proteins further comprise one or more proteins selected from the group consisting of C-type lectin domain family 4 member A (CLEC4A), fibroblast growth factor 21 (FGF21), trefoil factor 2 (TFF2), and hepatocyte growth factor (HGF). 
     
     
         40 . The method of any one of  claims 37 - 39 , wherein the four or more proteins comprise PlGF, sFlt1, KIM1, and CLEC4A. 
     
     
         41 . The method of any one of  claims 37 - 40 , wherein the plurality of different probes comprises probes with specific affinity to fibroblast growth factor 21 (FGF21), and the four or more proteins comprise FGF21. 
     
     
         42 . The method of any one of  claims 37 - 41 , wherein the plurality of different probes comprises probes with specific affinity for endoglin, and the four or more proteins comprise endoglin. 
     
     
         43 . The method of any one of  claims 37 - 42 , wherein the plurality of different probes comprises probes with specific affinity for decorin, and the four or more proteins comprise decorin. 
     
     
         44 . The method of any one of  claims 37 - 43 , wherein the plurality of different probes comprises probes with specific affinity for cluster of differentiation 274 (CD274), and the four or more proteins comprise CD274. 
     
     
         45 . The method of any one of  claims 37 - 44 , wherein the plurality of different probes comprises probes with specific affinity for hepatocyte growth factor (HGF), and the four or more proteins comprise HGF. 
     
     
         46 . The method of any one of  claims 37 - 45 , wherein the plurality of different probes comprises probes with specific affinity for trefoil factor 2 (TFF2), and the four or more proteins comprises TFF2. 
     
     
         47 . The method of any one of  claims 37 - 46 , wherein the plurality of different probes comprises probes with specific affinity for pappalysin-2 (PAPP-A2), and the four or more proteins comprises PAPP-A2. 
     
     
         48 . The method of any one of  claims 37 - 47 , wherein the biological sample has been collected from the pregnant human female after gestational week 20. 
     
     
         49 . The method of any one of  claims 37 - 48 , wherein the biological sample has been collected from the pregnant female prior to gestational week 30. 
     
     
         50 . The method of any one of  claims 37 - 49 , wherein the biological sample is a urine, blood, amniotic fluid, exosome, plasma, or serum sample. 
     
     
         51 . The method of any one of  claims 37 - 49 , wherein the biological sample is from blood of the pregnant human female. 
     
     
         52 . The method of any one of  claims 37 - 51 , wherein the amount or concentration of no more than 20, no more than 15, nor more than 10, nor more than 9, no more than 8, no more than 7, no more than 6, nor more than 5, or no more than 4 proteins is determined. 
     
     
         53 . The method of any one of  claims 37 - 52 , wherein one or more of the plurality of different probes are antibodies or antibody fragments. 
     
     
         54 . The method of any one of  claims 37 - 53 , wherein each of the plurality of different probes are antibodies or antibody fragments. 
     
     
         55 . The method of any one of  claims 37 - 54 , wherein the plurality of probes contact the biological sample or a fraction thereof in a single reaction vessel. 
     
     
         56 . The method of any one of  claims 37 - 54 , wherein the plurality of probes contact the biological sample or a fraction thereof in separate reaction vessels for each protein of the four or more proteins. 
     
     
         57 . The method of any one of  claims 37 - 56 , further comprising a second plurality of probes, wherein the second plurality of probes comprises a probe set that is specific for binding to PlGF, a probe set that is specific for binding to sFlt1, a probe set that is specific for binding to KIM1, and a probe set that is specific for binding to CLEC4A, wherein the second plurality of probes binds to its corresponding protein at an epitope that differs from the epitope to which each of the first plurality of probes binds. 
     
     
         58 . The method of  claim 57 , wherein coincident binding of at least one pair of the first and the second plurality of probes to the same protein molecules in the sample is detected by a luminescent oxygen channeling immunoassay (LOCI), a time-resolved fluorescence resonance energy transfer (TR-FRET) assay, an amplified luminescent proximity homogenous assay, an enzyme-linked immunosorbent assay, a proximity extension assay, or a lateral flow assay. 
     
     
         59 . A method for managing a pregnant human subject and identifying the pregnant human subject as not at risk for preeclampsia for a specified period of time, the method comprising:
 (a) identifying, via a test having (1) a specificity of greater than 80% and (2) a sensitivity of greater than 85%, the pregnant subject as not at risk for developing preeclampsia within the specified period of time, wherein the specified period of time is between one week and six weeks; and   (b) managing the pregnant human subject identified as not at risk for developing preeclampsia within the specified period of time by proceeding with ambulant monitoring treatment of said pregnant patient without treating the patient for preeclampsia.   
     
     
         60 . The method of  claim 59 , wherein the test has a specificity of at least 82.0%, at least 84.0%, at least 85.0%, at least 87.0%, at least 88.0%, at least 89.0%, at least 90.0%, at least 90.5%, at least 91.0%, or at least 91.5%. 
     
     
         61 . The method of  claim 59  or  60 , wherein the test has a sensitivity of at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%. 
     
     
         62 . The method of any one of  claims 59 - 61 , wherein the test has a negative predictive value of at least about 95.0%, 96.0%, 97.0%, 98.0%, 98.2%, 98.4%, 98.5%, 98.7%, 99%, 99.2%, or 99.5% when applied to a random population of ethnically diverse pregnant women after gestational week 20 that exhibit one or more of (1) high blood pressure and (2) proteinuria. 
     
     
         63 . The method of any one of  claims 59 - 62 , wherein the test has a positive predictive value of at least about 30%, at least about 32%, at least about 35%, at least about 37%; at least about 40%, at least about 42%, at least about 45%, at least about 50%, at least about 55%, or at least about 57% when applied to a random population of ethnically diverse pregnant women after gestational week 20 that exhibit one or more of (1) high blood pressure and (2) proteinuria. 
     
     
         64 . The method of any one of  claims 59 - 63 , wherein the negative predictive value of the test is higher than the positive predictive value of the test. 
     
     
         65 . The method of any one of  claims 59 - 63 , wherein the specified period of time is between one week and four weeks, one week and three weeks, or one week and two weeks. 
     
     
         66 . The method of any one of  claims 59 - 65 , wherein the test comprises determining an amount or concentration of each of four or more proteins selected from proteins listed in Table A or Table B. 
     
     
         67 . The method of  claim 66 , wherein the four or more proteins comprise PlGF, sFlt-1, KIM1, and CLEC4A. 
     
     
         68 . The method of  claim 67 , wherein the four or more proteins further comprise FGF21. 
     
     
         69 . A method of treating a pregnant human subject, the method comprising:
 obtaining an indicium generated, at least in part, from a determination of levels for four or more proteins listed in Table A or Table B; and   changing a clinical regimen for the pregnant human subject based, at least in part, on the obtained indicium.   
     
     
         70 . The method of  claim 69 , wherein obtaining the indicium comprises:
 determining the levels for PlGF, sFlt1, KIM1, and CLEC4A, wherein the levels are determined from binding of each of PlGF, sFlt1, KIM1, and CLEC4A to corresponding probes.   
     
     
         71 . The method of  claim 70 , wherein the obtaining the indicium further comprises determining the level for FGF21, wherein the level of FGF21 is determined from binding of FGF21 to corresponding probes. 
     
     
         72 . The method of any one of  claims 69 - 71 , further comprising classifying the pregnant subject as having a low risk of having preeclampsia or developing preeclampsia within a specified period of time. 
     
     
         73 . The method of  claim 72 , wherein the pregnant subject is classified by any method described herein. 
     
     
         74 . The method of any one or  claims 69 - 73 , further comprising administering an antihypertensive drug to the patient. 
     
     
         75 . The method of  claim 74 , wherein the antihypertensive drug is a central alpha agonist, a vasodilator, a calcium-channel blocker, an alpha-blocker or a beta-blocker. 
     
     
         76 . The method of  claim 74 , wherein the antihypertensive drug is methyldopa, labetalol, nifedipine, verapamil, clonidine, hydralazine, diazoxide, prazosin, or oxprenolol. 
     
     
         77 . A mixture comprising:
 a fluid sample from a pregnant female subject;   a first plurality of different probes, wherein the first plurality of different probes comprises different probes, each with specific affinity for four or more proteins selected from proteins listed in Table A or Table B.   
     
     
         78 . The mixture of  claim 77 , wherein the four or more proteins comprise:
 (a) placental growth factor (PlGF);   (b) one or more angiogenesis-associated proteins selected from the group consisting of soluble fms-like tyrosine kinase 1 (sFlt1), endoglin, pappalysin 2 (PAPP-A2), and decorin; and   (c) one or more kidney damage associated-proteins selected from the group consisting of (1) kidney injury molecule-1 (KIM1), (2) programmed cell death 1 ligand 1 (CD274), and decorin.   
     
     
         79 . The method of  claim 78 , wherein the four or more proteins further comprise one or more proteins selected from the group consisting of C-type lectin domain family 4 member A (CLEC4A), fibroblast growth factor 21 (FGF21), trefoil factor 2 (TFF2), and hepatocyte growth factor (HGF). 
     
     
         80 . The mixture of any one of  claims 77 - 79 , wherein the four or more proteins comprise PlGF, sFlt1, KIM1, and CLEC4A. 
     
     
         81 . The mixture of any one of  claims 77 - 80 , wherein the four or more proteins comprise FGF 21. 
     
     
         82 . The mixture of any one of  claims 77 - 81 , wherein the four or more proteins comprise endoglin. 
     
     
         83 . The mixture of any one of  claims 77 - 82 , wherein the four or more proteins comprise decorin. 
     
     
         84 . The mixture of any one of  claims 77 - 83 , wherein the four or more proteins comprise CD274. 
     
     
         85 . The mixture of any one of  claims 77 - 84 , wherein the four or more proteins comprise HGF. 
     
     
         86 . The mixture of any one of  claims 77 - 85 , wherein the four or more proteins comprise TFF2. 
     
     
         87 . The mixture of any one of  claims 77 - 86 , wherein the four or more proteins comprise PAPP-A2. 
     
     
         88 . The mixture of any one of  claims 77 - 87  wherein the fluid sample has been collected after gestational week 20. 
     
     
         89 . The mixture of any one of  claims 77 - 88 , wherein the fluid sample has been collected from the pregnant female prior to gestational week 30. 
     
     
         90 . The mixture of any one of  claims 77 - 89 , wherein the mixture includes no more than 20, no more than 16, no more than 14, no more than 12, no more than 10, no more than eight, no more than seven, no more than 6, no more than 5, or nor more than 4 sets of probes that are designed to bind to different proteins in the fluid sample. 
     
     
         91 . The mixture of any one of  claims 77 - 90 , wherein the fluid sample is from a blood, plasma, serum, or exosome sample. 
     
     
         92 . The method of any one of  claims 77 - 91 , wherein the fluid sample was obtained from the subject after the subject has shown one or more symptoms of preeclampsia, wherein the symptoms of preeclampsia are selected from (1) high blood pressure and (2) proteinuria. 
     
     
         93 . The method of  claim 77 - 92 , wherein the fluid sample was obtained from the subject after the subject has shown both (1) high blood pressure and (2) proteinuria. 
     
     
         94 . The method of any one of  claims 77 - 93 , wherein the first plurality of different probes comprises a first set of probes with specific affinity for each of the four or more proteins and a second set of probes with specific affinity for each of the four or more proteins, wherein each set of the two sets of probes binds to different epitopes. 
     
     
         95 . The mixture of  claim 94 , wherein the first set of probes and the second set of probes are conjugated to pairs of oligonucleotides containing complementary hybridization regions for each protein-specific probe pair. 
     
     
         96 . The mixture of  claim 94 , wherein the first set of probes and the second set of probes are conjugated to unique FRET pairs of fluorophores for each protein-specific probe pair. 
     
     
         97 . The mixture of  claim 94 , wherein, for each protein-specific probe pair, one probe is conjugated to biotin or a streptavidin-binding analog thereof. 
     
     
         98 . The mixture of  claim 94 , further comprising (a) a photosensitizer; and (b) an oxygen-sensitive dye, wherein one of (a) and (b) is capable of binding the first set of probes, and the other is capable of binding the second set of probes. 
     
     
         99 . The mixture of any one of  claims 77 - 98 , wherein one or more of the first plurality of different probes are antibodies or antibody fragments. 
     
     
         100 . The mixture of any one of  claims 77 - 99 , wherein each of the first plurality of different probes are antibodies or antibody fragments. 
     
     
         101 . A reaction plate comprising a plurality of reaction wells, wherein the plurality of reaction wells comprises:
 a first well comprising (1) a first portion of a biological sample from a pregnant human subject, wherein the biological sample was obtained from a pregnant human subject after 20 weeks of gestation, and (2) a first set of probes for binding to PlGF;   a second well comprising (1) a second portion of the biological sample from the pregnant human subject, and (2) a second set of probes for binding to sFlt1, endoglin, pappalysin 2 (PAPP-A2), or decorin;   a third well comprising (1) a third portion of the biological sample from the pregnant human subject, and (2) a third set of probes for binding to KIM1, (2) programmed cell death 1 ligand 1 (CD274), or decorin; and   a fourth well comprising (1) a fourth portion of the biological sample from the pregnant human subject, and (2) a fourth set of probes for binding to CLEC4A, FGF21, TFF2, or HGF.   
     
     
         102 . The reaction plate of  claim 101 , wherein the second set of probes in the second well are configured to bind to sFlt1. 
     
     
         103 . The reaction plate of  claim 101  or  claim 102 , wherein the third set of probes in the third well are configured to bind to KIM1. 
     
     
         104 . The reaction plate of any one of  claims 101 - 103 , wherein the fourth set of probes in the fourth well are configured to bind to CLEC4A. 
     
     
         105 . The reaction plate of any one of  claims 101 - 104 , wherein the plurality of wells comprises a fifth well, the fifth well comprising (1) a fifth portion of the biological sample from the pregnant human subject and (2) a fifth set of probes for binding to FGF21. 
     
     
         106 . The reaction plate of any one of  claims 101 - 105 , wherein the plurality of reaction wells comprises a well, the well comprising a portion of the biological sample and a set of probes for binding to endoglin. 
     
     
         107 . The reaction plate of any one of  claims 101 - 106 , wherein the plurality of reaction wells comprises a well, the well comprising a portion of the biological sample and a set of probes for binding to decorin. 
     
     
         108 . The reaction plate of any one of  claims 101 - 107 , wherein the plurality of reaction wells comprises a well, the well comprising a portion of the biological sample and a set of probes for binding to CD274. 
     
     
         109 . The reaction plate of any one of  claims 101 - 108 , wherein the plurality of reaction wells comprises a well, the well comprising a portion of the biological sample and a set of probes for binding to HGF. 
     
     
         110 . The reaction plate of any one of  claims 101 - 109 , wherein the plurality of reaction wells comprises a well, the well comprising a portion of the biological sample and a set of probes for binding to TFF2. 
     
     
         111 . The reaction plate of any one of  claims 101 - 110 , wherein the plurality of reaction wells comprises a well, the well comprising a portion of the biological sample and a set of probes for binding to PAPP-A2. 
     
     
         112 . The reaction plate of any one of  claims 101 - 111 , wherein the biological sample has been collected from the pregnant female prior to gestational week 30. 
     
     
         113 . The reaction plate of any one of  claims 101 - 112 , wherein the biological sample is from blood of the pregnant human female. 
     
     
         114 . The reaction plate of any one of  claims 101 - 113 , wherein the plurality of wells of the reaction include probes for specific binding to no more than 20, no more than 15, no more than 12, no more than 10, no more than 8, no more than 7, no more than 6, no more than 5, or no more than 4 proteins. 
     
     
         115 . The reaction plate of any one of  claims 101 - 114 , wherein the probes comprise antibodies or antibody fragments. 
     
     
         116 . The reaction plate of any one of  claims 101 - 115 , wherein the biological sample is from the subject after the subject has shown one or more symptoms of preeclampsia, wherein the symptoms of preeclampsia are selected from (1) high blood pressure and (2) proteinuria. 
     
     
         117 . The reaction plate of  claim 101 - 116 , wherein the sample is from the subject after the subject has shown both (1) high blood pressure and (2) proteinuria. 
     
     
         118 . The reaction plate of any one of  claims 101 - 117 , wherein each set of probes comprises probes that specifically bind to different epitopes. 
     
     
         119 . A kit for ruling out preeclampsia in a pregnant female subject, the kit comprising:
 (a) probes for determining the levels of four or more proteins selected from the proteins listed in Tables 6 and 7;   (b) wherein the kit is designed to measure the levels of no more than 20, no more than 15, no more than 10, nor more than 8, no more than 7, no more than 6, nor more than 5, or no more than 4 proteins.   
     
     
         120 . The kit of  claim 119 , wherein the four or more proteins comprises:
 (a) placental growth factor (PlGF);   (b) one or more angiogenesis-associated proteins selected from the group consisting of soluble fms-like tyrosine kinase 1 (sFlt1), endoglin, pappalysin 2 (PAPP-A2), and decorin; and   (c) one or more kidney damage associated-proteins selected from the group consisting of (1) kidney injury molecule-1 (KIM1), (2) programmed cell death 1 ligand 1 (CD274), and decorin.   
     
     
         121 . The kit of  claim 120 , wherein the four or more proteins comprise one or more proteins selected from the group consisting of C-type lectin domain family 4 member A (CLEC4A), fibroblast growth factor 21 (FGF21), trefoil factor 2 (TFF2), and hepatocyte growth factor (HGF). 
     
     
         122 . The kit of any one of  claims 119 - 121 , wherein the four or more proteins comprise PlGF, sFlt1, KIM1, and CLEC4A. 
     
     
         123 . The kit of any one of  claims 119 - 122 , wherein the four or more proteins comprise FGF21. 
     
     
         124 . The kit of any one of  claims 119 - 123 , wherein the four or more proteins comprise endoglin. 
     
     
         125 . The kit of any one of  claims 119 - 124 , wherein the four or more proteins comprise decorin. 
     
     
         126 . The kit of any one of  claims 119 - 125 , wherein the four or more proteins comprise CD274. 
     
     
         127 . The kit of any one of  claims 119 - 126 , wherein the four or more proteins comprise HGF. 
     
     
         128 . The kit of any one of  claims 119 - 127 , wherein the four or more proteins comprise TFF2. 
     
     
         129 . The kit of any one of  claims 119 - 128 , wherein the four or more proteins comprise PAPP-A2. 
     
     
         130 . The kit of any one of  claims 119 - 129 , further comprising instructions for carrying out an immunoassay. 
     
     
         131 . The kit of any one of  claims 119 - 130 , wherein the kit is an enzyme-linked immunosorbent assay kit. 
     
     
         132 . The kit of any one of  claims 119 - 131 , wherein one or more of the probes are attached to substrate. 
     
     
         133 . The kit of any one of  claims 119 - 132 , wherein the kit is for a lateral flow immunoassay. 
     
     
         134 . A system for ruling out preeclampsia in a pregnant female subject for a specified period of time, the system comprising:
 a processor;   an input module for inputting levels of at least four proteins in a biological sample, wherein the at least four proteins are selected from Tables 6 and 7;   a computer readable medium containing instructions that, when executed by the processor, perform a first algorithm on the input levels of the at least four proteins; and   an output module providing one or more indicia based on the input levels of the at least four proteins, wherein the one or more indicia are indicative of the subject not having preeclampsia for at least a specified period of time.   
     
     
         135 . The system of  claim 134 , further comprising probes to each the at least four proteins. 
     
     
         136 . The system of  claim 134  or  claim 135 , wherein the system is designed to output the one or more indicia based on input levels for no more than 15, no more than 10, no more than 8, no more than 7, nor more than 6, no more than 5, or no more than 4 proteins. 
     
     
         137 . The system of any one of  claims 134 - 136 , wherein the at least four proteins comprise:
 (a) placental growth factor (PlGF);   (b) one or more angiogenesis-associated proteins selected from the group consisting of soluble fms-like tyrosine kinase 1 (sFlt1), endoglin, pappalysin 2 (PAPP-A2), and decorin; and   (c) one or more kidney damage associated-proteins selected from the group consisting of (1) kidney injury molecule-1 (KIM1), (2) programmed cell death 1 ligand 1 (CD274), and decorin.   
     
     
         138 . The system of any one of  claims 134 - 137 , wherein the at least four proteins comprise one or more proteins selected from the group consisting of C-type lectin domain family 4 member A (CLEC4A), fibroblast growth factor 21 (FGF21), trefoil factor 2 (TFF2), and hepatocyte growth factor (HGF). 
     
     
         139 . The system of any one of  claims 134 - 138 , wherein the at least four proteins comprise PlGF, sFlt1, KIM1, and CLEC4A. 
     
     
         140 . The system of any one of  claims 134 - 139 , wherein the at least four proteins comprise FGF21. 
     
     
         141 . The system of any one of  claims 134 - 140 , wherein the at least four proteins comprise decorin. 
     
     
         142 . The system of any one of  claims 134 - 141 , wherein the at least four proteins comprise CD274. 
     
     
         143 . The system of any one of  claims 134 - 142 , wherein the at least four proteins comprise HGF. 
     
     
         144 . The system of any one of  claims 134 - 143 , wherein the at least four proteins comprise TFF2. 
     
     
         145 . The system of any one of  claims 134 - 144 , wherein the at least four proteins comprise PAPP-A2. 
     
     
         146 . The system of any one of  claims 134 - 145 , wherein the algorithm comprises a correction based on gestational age. 
     
     
         147 . The system of any one of  claims 134 - 146 , wherein the algorithm comprises a logistics regression. A method for classifying a pregnant human subject as not having and/or not going to develop preeclampsia within a specified time period, the method comprising:
 obtaining a sample from a pregnant human subject who has been identified as having one or more symptoms or risk factors for preeclampsia;   running a test to obtain a protein expression profile, wherein the protein expression profile includes levels of two or more proteins from Tables A and B;   applying a classifier algorithm to the expression profile, wherein the classifier algorithm calculates an index; and   comparing the index to a reference value to determine whether to classify the pregnant human subject as not having and/or not going to develop preeclampsia within the specified time period.   
     
     
         148 . The method of  claim 147 , wherein the test has:
 a specificity of at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99%; and   a sensitivity of at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99%.   
     
     
         149 . The method of  claim 147  or  claim 148 , wherein the one or more symptoms comprises one or both of high blood pressure and proteinuria. 
     
     
         150 . The method of any one of  claims 147 - 149 , wherein the specified period of time is between one and six weeks (e.g., between one and four weeks). 
     
     
         151 . The method of any one of  claims 147 - 150 , wherein the protein expression profile includes levels of three or more proteins from Tables A and B. 
     
     
         152 . The method of any one of  claims 147 - 151 , wherein the protein expression profile includes levels of four or more proteins from Tables A and B. 
     
     
         153 . The method of any one of  claims 147 - 152 , wherein the protein expression profile includes levels of five or more proteins from Tables A and B. 
     
     
         154 . The method of any one of  claims 147 - 153 , wherein the expression profiles of no more than 15, 10, 8, or 6 proteins are determined.