US 20150293131A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0293131 A1 Anderberg et al. (43) Pub. Date: Oct. 15, 2015

(54) METHODS AND COMPOSITIONS FOR Publication Classification DAGNOSIS AND PROGNOSIS OF SEPSIS (51) Int. Cl. (71) Applicant: ASTUTE MEDICAL, INC. San GOIN33/92 (2006.01) Diego, CA (US) GOIN33/74 (2006.01) (72) Inventors: Joseph Anderberg, Encinitas, CA (US); GOIN 33/573 (2006.01) Jeff Gray, Solana Beach, CA (US); Paul GOIN33/68 (2006.01) McPherson, Encinitas, CA (US); Kevin (52) U.S. Cl. Nakamura, Cardiff by the Sea, CA CPC ...... G0IN33/92 (2013.01); G0IN33/6893 (US); James Patrick Kampf, San (2013.01); G0IN33/6869 (2013.01); G0IN Diego, CA (US) 33/74 (2013.01); G0IN33/6866 (2013.01); (73) Assignee: ASTUTE MEDICAL, INC., San Diego, G0IN33/573 (2013.01); G0IN33/6863 CA (US) (2013.01); G0IN 2800/26 (2013.01); G0IN 2800/52 (2013.01); G0IN 2800/50 (2013.01) (21) Appl. No.: 14/390,338

(86). PCT No.: PCT/US2013/035032 S371 (c)(1), The present invention relates to methods and compositions (2) Date: Oct. 2, 2014 for monitoring, diagnosis, prognosis, and determination of O O treatment regimens in Sepsis patients and in patients at risk for Related U.S. Application Data sepsis. In particular, the invention relates to using assays that (60) Provisional application No. 61/619,037, filed on Apr. detect one or more biomarkers as diagnostic and prognostic 2, 2012. biomarker assays in Such patients. US 2015/0293131 A1 Oct. 15, 2015

METHODS AND COMPOSITIONS FOR ment of acute kidney injury (AKI) during sepsis increases DAGNOSIS AND PROGNOSS OF SEPSIS patient morbidity, predicts higher mortality, has a significant effect on multiple organ functions, is associated with an 0001. This application claims priority to U.S. Provisional increased length of stay in the intensive care unit, and hence Patent Application 61/619,037 filed Apr. 2, 2012, which is consumes considerable healthcare resources. A biomarker hereby incorporated by reference in its entirety including all able to stratify risk during the first days of admission could tables, figures, and claims. differ from one that provides a prediction later in the course of disease. A sequential determination of a biomarker may also BACKGROUND OF THE INVENTION be of use in following the acute response to treatment in 0002 The following discussion of the background of the patients with sepsis. invention is merely provided to aid the reader in understand 0007. There remains in the art methods and compositions ing the invention and is not admitted to describe or constitute for evaluating sepsis in patients in order to identify onset of prior art to the present invention. disease, and those most at risk for poor outcomes. 0003. The term “sepsis' has been used to describe a vari ety of clinical conditions related to systemic manifestations BRIEF SUMMARY OF THE INVENTION of inflammation accompanied by an infection. Because of 0008. It is an object of the invention to provide methods clinical similarities to inflammatory responses secondary to and compositions for evaluating a sepsis patient. As described non-infectious etiologies, identifying sepsis has been a par herein, measurement of one or more biomarkers selected ticularly challenging diagnostic problem. Recently, the from the group consisting of Alpha-2 macroglobulin, Angio American College of Chest Physicians and the American genin, -1 receptor, Angiopoietin-related Society of Critical Care Medicine (Bone et al., Chest 101: 3, Angiopoietin-related protein 4, Angiopoietin-related pro 1644-53, 1992) published definitions for "Systemic Inflam tein 6, Apollipoprotein A-II, Apollipoprotein C-III, Apollipo matory Response Syndrome' (or “SIRS), which refers gen protein(a), Bone morphogenetic protein 7. Cadherin-16, Cad erally to a severe systemic response to an infectious or non herin-3, Cancer Antigen 15-3, Cancer Antigen 19-9., infectious insult, and for the related syndromes “sepsis.” Carbonic anhydrase 9, Carcinoembryonic antigen-related "severe sepsis, and 'septic shock, and extending to multiple cell adhesion molecule 1, Carcinoembryonic antigen-related organ dysfunction syndrome (“MODS). These definitions, cell adhesion molecule 5, Caspase-3, active, Cathepsin S. described below, are intended for each of these phrases for the C C motif chemokine 1, C-C motif chemokine 15, C C purposes of the present application. motif chemokine 17, C C motif chemokine 18, C C motif 0004. A systemic inflammatory response leading to a chemokine 20, C-C motif chemokine 21, C-C motif diagnosis of SIRS may be related to both infection and to chemokine 24, Clusterin, Collagenase 3, C-peptide (), numerous non-infective etiologies, including burns, pancre C X—C motif chemokine 11, C X—C motif chemokine atitis, trauma, heat stroke, and neoplasia. While conceptually 16, Cyclin-dependent kinase inhibitor 1. DDRGK domain it may be relatively simple to distinguish between sepsis and containing protein 1, Endostatin, Epidermal non-septic SIRS, no diagnostic tools have been described to receptor, Epiregulin, Epithelial cell adhesion molecule, unambiguously distinguish these related conditions. See, receptor, 19, Fibro e.g., Llewelyn and Cohen, Int. Care Med. 27: S10-S32, 2001. blast growth factor 23, Galectin-3, Gastric inhibitory For example, because more than 90% of sepsis cases involve polypeptide, Glucagon, Glucagon-like peptide 1. Glutathione bacterial infection, the “gold standard for confirming infec S-transferase P. Heat shock protein beta-1, Heparin-binding tion has been microbial growth from blood, urine, pleural growth factor 1, Hepatitis A virus cellular receptor 1, Hepa fluid, cerebrospinal fluid, peritoneal fluid, Synnovial fluid, tocyte , Insulin, Insulin-like growth sputum, or other tissue specimens. Such culture has been factor 1 receptor, Insulin-like growth factor-binding protein reported, however, to fail to confirm 50% or more of patients 6, Insulin-like growth factor-binding protein 7. Interferon exhibiting strong clinical evidence of sepsis. See, e.g., Jaimes alpha-2, -20, Interleukin-21, Interleukin-28A, et al., Int. Care Med 29: 1368-71, published electronically Interleukin-29, Interleukin-33, Involucrin, Islet amyloid Jun. 26, 2003. polypeptide, Keratin, type II cytoskeletal 1 (Keratin-1, -10 0005 Thus, despite improvements in the management of mix), Lymphatic vessel endothelial hyaluronic acid receptor critically ill patients, sepsis remains the leading cause of 1, Macrophage metalloelastase, Matrix metalloproteinase-9: death in Such patients. This makes early determination diag Metalloproteinase inhibitor 2 complex, Metalloproteinase nosis vital. The two biomarkers that have been most widely inhibitor 3, Myeloid differentiation primary response protein studied and used in patients with sepsis are C-reactive protein MyD88, Netrin-1, Netrin-4, Neuronal cell adhesion mol (CRP) and procalcitonin (PCT). Levels of both these biom ecule, NF-kappa-B inhibitor alpha, Nidogen-1, Osteocalcin, arkers have been demonstrated to be raised in patients with Pappalysin-1, Poly ADP-ribose polymerase 1 (cleaved), sepsis, but because they lack specificity for sepsis and levels Probetacellulin, Prostate-specific antigen, Prostatic acid may be raised in other inflammatory diseases, these biomar phosphatase, Protein NOV homolog, Protransforming kers are more useful for ruling out sepsis than for ruling it in, growth factor alpha, P-selectin glycoprotein ligand 1, Sex that is, a completely normal value makes a diagnosis of sepsis hormone-binding globulin, SL cytokine, Tenascin, Thrombo less likely, but an elevated level may not be due to infection. spondin-2, Thymic stromal lymphopoietin, Transmembrane 0006 Moreover, biomarkers are relevant in clinical prac glycoprotein NMB, Trefoil factor 3, Tubulointerstitial tice not only for their ability to diagnose a pathological con nephritis antigen, Tumor necrosis factor receptor Superfamily dition, but also for predicting morbidity and outcome. The member 10B, Tumor necrosis factor receptor superfamily ability to assigna severity of illness and outcome likelihood to member 8, Versican core protein, Bone morphogenetic pro a sepsis patient is equally vital for triaging of patients and tein 7, Carbonic anhydrase 9, Caspase-9, Collagenase 3, guiding therapeutic decisions. By way of example, develop Granzyme M. Heparin-binding EGF-like growth factor. Insu US 2015/0293131 A1 Oct. 15, 2015 lin receptor substrate 1, Keratin, type II cytoskeletal 6 (6A, nephritis antigen, Tumor necrosis factor receptor Superfamily -6B, -6C mix), Myeloid differentiation primary response pro member 10B, Tumor necrosis factor receptor superfamily tein MyD88, SL cytokine, Vascular endothelial growth factor member 8, Versican core protein, Bone morphogenetic pro D.Vascular endothelial growth factor receptor 2, and Vascular tein 7, Carbonic anhydrase 9, Caspase-9, Collagenase 3, endothelial growth factor receptor 3 (each referred to herein Granzyme M. Heparin-binding EGF-like growth factor. Insu as a “sepsis biomarker) can be used for diagnosis, prognosis, lin receptor substrate 1, Keratin, type II cytoskeletal 6 (6A, risk stratification, staging, monitoring, categorizing and -6B, -6C mix), Myeloid differentiation primary response pro determination of further diagnosis and treatment regimens in tein MyD88, SL cytokine, Vascular endothelial growth factor sepsis patients. D.Vascular endothelial growth factor receptor 2, and Vascular 0009. The sepsis biomarkers of the present invention may endothelial growth factor receptor 3, the results of which be used, individually or in panels comprising a plurality of assay(s) is/are then correlated to the status of the patient. This sepsis biomarkers, for identifying a subject Suffering from correlation to status may include correlating the assay result SIRS, sepsis, severe sepsis, septic shock and/or MODS, for (s) to one or more of diagnosis, risk stratification, prognosis, distinguishing amongst these conditions, for assigning a risk staging, classifying and monitoring of the sepsis patient as that a subject at risk for sepsis will progress to sepsis, severe described herein. Thus, the present invention utilizes one or sepsis, septic shock and/or MODS; or for assigning a prog more sepsis biomarkers of the present invention for the evalu nosis to a subject Suffering from one or more of these condi ation of a patient. tions, etc. 0011. In certain embodiments, the methods for evaluating 0010. In a first aspect, the present invention relates to a sepsis patient described herein are methods for risk strati methods for evaluating a sepsis patient or a patient being fication of the sepsis patient; that is, assigning a likelihood of evaluated for a possible sepsis diagnosis. These methods one or more future changes in health status to the sepsis comprise performing an assay method that is configured to patient. In these embodiments, the assay result(s) is/are cor detect one or more biomarkers selected from the group con related to one or more Such future changes. The following are sisting of Alpha-2 macroglobulin, Angiogenin, Angiopoi preferred risk stratification embodiments. etin-1 receptor, Angiopoietin-related protein 3, Angiopoietin 0012. In preferred risk stratification embodiments, these related protein 4, Angiopoietin-related protein 6. methods comprise determining a sepsis patient’s risk for Apollipoprotein A-II, Apollipoprotein C-III, Apollipoprotein future progression to a worsening (or improving) stage within (a), Bone morphogenetic protein 7, Cadherin-16, Cadherin-3, the definition of SIRS. By way of example, the method may Cancer Antigen 15-3, Cancer Antigen 19-9, Carbonic anhy comprise assigning a likelihood of progression from SIRS to drase 9, Carcinoembryonic antigen-related cell adhesion sepsis; from sepsis to severe sepsis; from Sepsis or severe molecule 1, Carcinoembryonic antigen-related cell adhesion sepsis to septic shock; from sepsis, severe sepsis, or septic molecule 5, Caspase-3, active, Cathepsin S. C C motif shock to MODS. Alternatively, the method may comprise chemokine 1, C-C motif chemokine 15, C-C motif assigning a likelihood of progression from recovery from chemokine 17, C-C motif chemokine 18, C-C motif sepsis; from severe sepsis; from septic shock; from MODS. chemokine 20, C-C motif chemokine 21, C-C motif For example, the measured concentration(s) may each be chemokine 24, Clusterin, Collagenase 3, C-peptide (Insulin), compared to a threshold value. For a positive going sepsis C X—C motif chemokine 11, C X-C motif chemokine biomarker, an increased likelihood of progression to a wors 16, Cyclin-dependent kinase inhibitor 1. DDRGK domain ening stage is assigned to the sepsis patient when the mea containing protein 1, Endostatin, sured concentration is above the threshold, relative to a like receptor, Epiregulin, Epithelial cell adhesion molecule, lihood assigned when the measured concentration is below , Fibroblast growth factor 19, Fibro the threshold. For a “negative going sepsis biomarker, an blast growth factor 23, Galectin-3, Gastric inhibitory increased likelihood of progressing to a worsening stage is polypeptide, Glucagon, Glucagon-like peptide 1. Glutathione assigned to the sepsis patient when the measured concentra S-transferase P, Heat shock protein beta-1, Heparin-binding tion is below the threshold, relative to a likelihood assigned growth factor 1, Hepatitis A virus cellular receptor 1, Hepa when the measured concentration is above the threshold. tocyte growth factor receptor, Insulin, Insulin-like growth 0013. In other preferred risk stratification embodiments, factor 1 receptor, Insulin-like growth factor-binding protein these methods comprise determining a sepsis patient's risk 6, Insulin-like growth factor-binding protein 7. Interferon for future reduced renal function, and the assay result(s) is/are alpha-2, Interleukin-20, Interleukin-21, Interleukin-28A, correlated to a likelihood of such reduced renal function. For Interleukin-29, Interleukin-33, Involucrin, Islet amyloid example, the measured concentrations may each be compared polypeptide, Keratin, type II cytoskeletal 1 (Keratin-1, -10 to a threshold value. For a "positive going sepsis biomarker, mix), Lymphatic vessel endothelial hyaluronic acid receptor an increased likelihood of suffering a future reduced renal 1, Macrophage metalloelastase, Matrix metalloproteinase-9: function is assigned to the sepsis patient when the measured Metalloproteinase inhibitor 2 complex, Metalloproteinase concentration is above the threshold, relative to a likelihood inhibitor 3, Myeloid differentiation primary response protein assigned when the measured concentration is below the MyD88, Netrin-1, Netrin-4, Neuronal cell adhesion mol threshold. For a “negative going sepsis biomarker, an ecule, NF-kappa-B inhibitor alpha, Nidogen-1, Osteocalcin, increased likelihood of future reduced renal function is Pappalysin-1, Poly ADP-ribose polymerase 1 (cleaved), assigned to the sepsis patient when the measured concentra Probetacellulin, Prostate-specific antigen, Prostatic acid tion is below the threshold, relative to a likelihood assigned phosphatase, Protein NOV homolog, Protransforming when the measured concentration is above the threshold. growth factor alpha, P-selectin glycoprotein ligand 1, Sex 0014. In yet other preferred risk stratification embodi hormone-binding globulin, SL cytokine, Tenascin, Thrombo ments, these methods comprise determining a sepsis patients spondin-2, Thymic stromal lymphopoietin, Transmembrane risk for progression to ARF, and the result(s) is/are correlated glycoprotein NMB, Trefoil factor 3, Tubulointerstitial to a likelihood of such progression to ARF. For example, the US 2015/0293131 A1 Oct. 15, 2015

measured concentration(s) may each be compared to a thresh cellular receptor 1, receptor, Insu old value. For a "positive going sepsis biomarker, an lin, Insulin-like growth factor 1 receptor, Insulin-like growth increased likelihood of progression to ARF is assigned to the factor-binding protein 6. Insulin-like growth factor-binding sepsis patient when the measured concentration is above the protein 7. Interferon alpha-2, Interleukin-20. Interleukin-21, threshold, relative to a likelihood assigned when the mea Interleukin-28A, Interleukin-29, Interleukin-33, Involucrin, sured concentration is below the threshold. For a “negative Islet amyloid polypeptide, Keratin, type II cytoskeletal 1 going sepsis biomarker, an increased likelihood of progres (Keratin-1, -10 mix), Lymphatic vessel endothelial hyalu sion to ARF is assigned to the sepsis patient when the mea ronic acid receptor 1, Macrophage metalloelastase, Matrix sured concentration is below the threshold, relative to a like metalloproteinase-9:Metalloproteinase inhibitor 2 complex, lihood assigned when the measured concentration is above Metalloproteinase inhibitor 3, Myeloid differentiation pri the threshold. mary response protein MyD88, Netrin-1, Netrin-4, Neuronal 0015. And in other preferred risk stratification embodi cell adhesion molecule, NF-kappa-B inhibitor alpha, ments, these methods comprise determining a sepsis patients Nidogen-1, Osteocalcin, Pappalysin-1, Poly ADP-ribose outcome risk, and the assay result(s) is/are correlated to a polymerase 1 (cleaved), Probetacellulin, Prostate-specific likelihood mortality by the sepsis patient. For example, the antigen, Prostatic acid phosphatase, Protein NOV homolog, measured concentration(s) may each be compared to a thresh Protransforming growth factor alpha, P-selectinglycoprotein old value. For a "positive going sepsis biomarker, an ligand 1, Sex hormone-binding globulin, SL cytokine, Tena increased likelihood of mortality is assigned to the sepsis Scin, Thrombospondin-2, Thymic stromal lymphopoietin, patient when the measured concentration is above the thresh Transmembrane glycoprotein NMB, Trefoil factor 3, Tubu old, relative to a likelihood assigned when the measured lointerstitial nephritis antigen, Tumor necrosis factor receptor concentration is below the threshold. For a “negative going superfamily member 10B, Tumor necrosis factor receptor sepsis biomarker, an increased likelihood of mortality is superfamily member 8, Versican core protein, Bone morpho assigned to the sepsis patient when the measured concentra genetic protein 7, Carbonic anhydrase 9, Caspase-9, Colla tion is below the threshold, relative to a likelihood assigned genase 3, Granzyme M. Heparin-binding EGF-like growth when the measured concentration is above the threshold. factor, Insulin receptor Substrate 1, Keratin, type II cytoskel 0016. In such risk stratification embodiments, preferably etal 6 (6A, -6B, -6C mix), Myeloid differentiation primary the likelihood or risk assigned is that an event of interest is response protein MyD88, SL cytokine, Vascular endothelial more or less likely to occur within 180 days of the time at growth factor D. Vascular endothelial growth factor receptor which the body fluid sample is obtained from the sepsis 2, and Vascular endothelial growth factor receptor 3 is/are patient. In particularly preferred embodiments, the likelihood correlated to the occurrence or nonoccurrence disease. The orrisk assigned relates to an event of interest occurring within following are preferred diagnostic embodiments. In various a shorter time period such as 18 months, 120 days, 90 days, 60 embodiments, the methods comprise relating the assay result days, 45 days, 30 days, 21 days, 14 days, 7 days, 5 days, 96 (s) to ruling in or out one or more of the following diagnoses: hours, 72 hours, 48 hours, 36 hours, 24 hours, 12 hours, or that the Subject has at least sepsis; that the Subject has at least less. A risk at 0 hours of the time at which the body fluid severe sepsis; that the Subject has at least septic shock; that the sample is obtained from the sepsis patient is equivalent to subject has MODS. diagnosis of a current condition. 0018. In preferred diagnostic embodiments, these meth 0017. In other embodiments, the methods for evaluating ods comprise distinguishing among SIRS, sepsis, severe sep status described herein are methods for diagnosis, which sis, septic shock and/or MODS. These methods comprise refers to identifying a Subject Suffering from sepsis, severe relating the assay result(s) to ruling in or out one or more of sepsis, septic shock and/or MODS. In these embodiments, the the following diagnoses: that the subject has SIRS, but not assay result(s), for example measured concentration(s) of one sepsis, severe sepsis, septic shock, or MODS; that the subject or more biomarkers selected from the group consisting of has sepsis, but not severe sepsis, septic shock, or MODS; that Alpha-2 macroglobulin, Angiogenin, Angiopoietin-1 recep the subject has septic shock but not MODS; that the subject tor, Angiopoietin-related protein 3, Angiopoietin-related pro has MODS. For a positive going marker, an increased likeli tein 4, Angiopoietin-related protein 6, Apollipoprotein A-II, hood of the occurrence of a diagnosis is assigned to the patient Apollipoprotein C-III, Apollipoprotein(a), Bone morphoge when the measured concentration is above the threshold (rela netic protein 7, Cadherin-16, Cadherin-3, Cancer Antigen tive to the likelihood assigned when the measured concentra 15-3, Cancer Antigen 19-9, Carbonic anhydrase 9, Carcino tion is below the threshold); alternatively, when the measured embryonic antigen-related cell adhesion molecule 1, Carci concentration is below the threshold, an increased likelihood noembryonic antigen-related cell adhesion molecule 5. of the nonoccurrence of a diagnosis may be assigned to the Caspase-3, active, Cathepsin S, C-C motif chemokine 1, patient (relative to the likelihood assigned when the measured C C motif chemokine 15, C-C motif chemokine 17, C C concentration is above the threshold). For a negative going motif chemokine 18, C-C motif chemokine 20, C C motif marker, an increased likelihood of the occurrence of a diag chemokine 21, C-C motif chemokine 24, Clusterin, Colla nosis is assigned to the patient when the measured concen genase 3, C-peptide (Insulin), C X-C motif chemokine tration is below the threshold (relative to the likelihood 11, C X-C motif chemokine 16, Cyclin-dependent kinase assigned when the measured concentration is above the inhibitor 1, DDRGK domain-containing protein 1, Endosta threshold); alternatively, when the measured concentration is tin, Epidermal growth factor receptor, Epiregulin, Epithelial above the threshold, an increased likelihood of the nonoccur cell adhesion molecule, Erythropoietin receptor, Fibroblast rence of a diagnosis may be assigned to the patient (relative to growth factor 19, Fibroblast growth factor 23, Galectin-3, the likelihood assigned when the measured concentration is Gastric inhibitory polypeptide, Glucagon, Glucagon-like below the threshold). peptide 1. Glutathione S-transferase P. Heat shock protein 0019. A variety of methods may be used by the skilled beta-1, Heparin-binding growth factor 1, Hepatitis A virus artisan to arrive at a desired threshold value for use in these US 2015/0293131 A1 Oct. 15, 2015

methods. For example, for a positive going marker the thresh least about 0.8, even more preferably at least about 0.9 and old value may be determined from a population of SIRS most preferably at least about 0.95, with a corresponding patients not having sepsis by selecting a concentration repre sensitivity greater than 0.2, preferably greater than about 0.3, senting the 75'., 85,90", 95", or 99 percentile of a sepsis more preferably greater than about 0.4, still more preferably biomarker measured in such SIRS patients. Alternatively, the at least about 0.5, even more preferably about 0.6, yet more threshold value may be determined from a “diseased popu preferably greater than about 0.7, still more preferably greater lation of sepsis patients by selecting a concentration repre than about 0.8, more preferably greater than about 0.9, and senting the 75", 85",90", 95", or 99" percentile of a sepsis most preferably greater than about 0.95: biomarker measured in Such sepsis patients. a sensitivity of greater than 0.5, preferably at least about 0.6, 0020. Alternatively, the threshold value may be deter more preferably at least about 0.7, still more preferably at mined from a “diseased population of sepsis patients having least about 0.8, even more preferably at least about 0.9 and a predisposition for an outcome such as death, worsening most preferably at least about 0.95, with a corresponding disease, AKI, etc.), by selecting a concentration representing specificity greater than 0.2, preferably greater than about 0.3, the 75'., 85,90", 95", or 99" percentile of a sepsis biom more preferably greater than about 0.4, still more preferably arker measured in Such sepsis patients. In another alternative, at least about 0.5, even more preferably about 0.6, yet more the threshold value may be determined from a prior measure preferably greater than about 0.7, still more preferably greater ment of a sepsis biomarker in the same sepsis patient; that is, than about 0.8, more preferably greater than about 0.9, and a temporal change in the level of a sepsis biomarker in the most preferably greater than about 0.95: sepsis patient may be used to assign risk to the sepsis patient. at least about 75% sensitivity, combined with at least about 0021. The foregoing discussion is not meant to imply, 75% specificity: however, that the sepsis biomarkers of the present invention a positive likelihood ratio (calculated as sensitivity/(1-speci must be compared to corresponding individual thresholds. ficity)) of greater than 1, at least about 2, more preferably at Methods for combining assay results can comprise the use of least about 3, still more preferably at least about 5, and most multivariate logistical regression, loglinear modeling, neural preferably at least about 10; or network analysis, n-of-manalysis, decision tree analysis, cal a negative likelihood ratio (calculated as (1-sensitivity)/ culating ratios of markers, etc. This list is not meant to be specificity) of less than 1, less than or equal to about 0.5, more limiting. In these methods, a composite result which is deter preferably less than or equal to about 0.3, and most preferably mined by combining individual markers may be treated as if less than or equal to about 0.1. it is itself a marker; that is, a threshold may be determined for 0024. The term “about” in the context of any of the above the composite result as described herein for individual mark measurements refers to +/-5% of a given measurement. ers, and the composite result for an individual patient com 0025 Multiple thresholds may also be used to assess a pared to this threshold. sepsis patient. For example, a “first subpopulation identified 0022. The ability of a particular test to distinguish two by an existing disease, predisposition to a future outcome for populations can be established using ROC analysis. For the sepsis patient, predisposition to mortality, etc., and a example, ROC curves established from a “first subpopula 'second Subpopulation which is not so predisposed can be tion which has a particular disease (or which is predisposed to combined into a single group. This group is then Subdivided Some outcome), and a 'second Subpopulation which does into three or more equal parts (known as tertiles, quartiles, not have the disease (or is not so predisposed) can be used to quintiles, etc., depending on the number of Subdivisions). An calculate a ROC curve, and the area under the curve provides odds ratio is assigned to sepsis patients based on which Sub a measure of the quality of the test. Preferably, the tests division they fall into. If one considers a tertile, the lowest or described herein provide a ROC curve area greater than 0.5, highest tertile can be used as a reference for comparison of the preferably at least 0.6, more preferably 0.7, still more prefer other Subdivisions. This reference Subdivision is assigned an ably at least 0.8, even more preferably at least 0.9, and most odds ratio of 1. The second tertile is assigned an odds ratio preferably at least 0.95. that is relative to that first tertile. That is, someone in the 0023. In certain aspects, the measured concentration of second tertile might be 3 times more likely to suffer one or one or more sepsis biomarkers, or a composite of such mark more future changes in disease status in comparison to some ers, may be treated as continuous variables. For example, any one in the first tertile. The third tertile is also assigned an odds particular concentration can be converted into a correspond ratio that is relative to that first tertile. ing probability of existing disease, of a future outcome for the 0026. In certain embodiments, the assay method is an sepsis patient, or mortality, of a SIRS classification, etc. In yet immunoassay. Antibodies for use in Such assays will specifi another alternative, a threshold that can provide an acceptable cally bind a full length sepsis biomarker of interest, and may level of specificity and sensitivity in separating a population also bind one or more polypeptides that are “related thereto, of sepsis patients into “bins' such as a “first subpopulation as that term is defined hereinafter. Numerous immunoassay and a “second subpopulation. A threshold value is selected formats are known to those of skill in the art. Preferred body to separate this first and second population by one or more of fluid samples are selected from the group consisting of urine, the following measures of test accuracy: blood, serum, saliva, tears, and plasma. an odds ratio greater than 1, preferably at least about 2 or more 0027. The foregoing method steps should not be inter or about 0.5 or less, more preferably at least about 3 or more preted to mean that the sepsis biomarker assay result(s) is/are or about 0.33 or less, still more preferably at least about 4 or used in isolation in the methods described herein. Rather, more or about 0.25 or less, even more preferably at least about additional variables or other clinical indicia may be included 5 or more or about 0.2 or less, and most preferably at least in the methods described herein. For example, a risk stratifi about 10 or more or about 0.1 or less; cation, diagnostic, classification, monitoring, etc. method a specificity of greater than 0.5, preferably at least about 0.6, may combine the assay result(s) with one or more variables more preferably at least about 0.7, still more preferably at measured for the sepsis patient selected from the group con US 2015/0293131 A1 Oct. 15, 2015

sisting of demographic information (e.g., weight, sex, age, DETAILED DESCRIPTION OF THE INVENTION race), medical history (e.g., family history, type of Surgery, pre-existing disease such as aneurism, congestive heart fail 0033. The present invention relates to methods and com ure, preeclampsia, eclampsia, diabetes mellitus, hyperten positions for diagnosis, differential diagnosis, risk stratifica Sion, coronary artery disease, proteinuria, or renal insuffi tion, monitoring, classifying and determination of treatment ciency, clinical variables (e.g., blood pressure, temperature, regimens in patients diagnosed with, or at risk of sepsis. In respiration rate), risk scores (APACHE score, PREDICT various embodiments, a measured concentration of one or score, TIMI Risk Score for UA/NSTEMI, Framingham Risk more biomarkers selected from the group consisting of Score). Alpha-2 macroglobulin, Angiogenin, Angiopoietin-1 recep 0028. When more than one marker is measured, the indi tor, Angiopoietin-related protein 3, Angiopoietin-related pro vidual markers may be measured in Samples obtained at the tein 4, Angiopoietin-related protein 6, Apollipoprotein A-II, same time, or may be determined from samples obtained at Apollipoprotein C-III, Apollipoprotein(a), Bone morphoge different (e.g., an earlier or later) times. The individual mark netic protein 7, Cadherin-16, Cadherin-3, Cancer Antigen ers may also be measured on the same or different body fluid 15-3, Cancer Antigen 19-9, Carbonic anhydrase 9, Carcino samples. For example, one sepsis biomarker may be mea embryonic antigen-related cell adhesion molecule 1, Carci Sured in a serum or plasma sample and another sepsis biom noembryonic antigen-related cell adhesion molecule 5. arker may be measured in a urine sample. In addition, assign Caspase-3, active, Cathepsin S, C-C motif chemokine 1, ment of a likelihood may combine an individual sepsis C C motif chemokine 15, C-C motif chemokine 17, C C biomarker assay result with temporal changes in one or more motif chemokine 18, C-C motif chemokine 20, C-C motif additional variables. chemokine 21, C-C motif chemokine 24, Clusterin, Colla 0029. In various related aspects, the present invention also genase 3, C-peptide (Insulin), C X-C motif chemokine relates to devices and kits for performing the methods 11, C X-C motif chemokine 16, Cyclin-dependent kinase described herein. Suitable kits comprise reagents sufficient inhibitor 1, DDRGK domain-containing protein 1, Endosta for performing an assay for at least one of the described sepsis tin, Epidermal growth factor receptor, Epiregulin, Epithelial biomarkers, together with instructions for performing the cell adhesion molecule, Erythropoietin receptor, Fibroblast described threshold comparisons. growth factor 19, Fibroblast growth factor 23, Galectin-3, 0030. In certain embodiments, reagents for performing Gastric inhibitory polypeptide, Glucagon, Glucagon-like Such assays are provided in an assay device, and Such assay peptide 1. Glutathione S-transferase P. Heat shock protein devices may be included in such a . Preferred reagents can beta-1, Heparin-binding growth factor 1, Hepatitis A virus comprise one or more solid phase antibodies, the Solid phase cellular receptor 1, Hepatocyte growth factor receptor, Insu antibody comprising antibody that detects the intended biom lin, Insulin-like growth factor 1 receptor, Insulin-like growth arker target(s) bound to a solid Support. In the case of sand factor-binding protein 6. Insulin-like growth factor-binding wich immunoassays, such reagents can also include one or protein 7. Interferon alpha-2, Interleukin-20. Interleukin-21, more detectably labeled antibodies, the detectably labeled Interleukin-28A, Interleukin-29, Interleukin-33, Involucrin, antibody comprising antibody that detects the intended biom Islet amyloid polypeptide, Keratin, type II cytoskeletal 1 arker target(s) bound to a detectable label. Additional (Keratin-1, -10 mix), Lymphatic vessel endothelial hyalu optional elements that may be provided as part of an assay ronic acid receptor 1, Macrophage metalloelastase, Matrix device are described hereinafter. metalloproteinase-9:Metalloproteinase inhibitor 2 complex, 0031 Detectable labels may include molecules that are Metalloproteinase inhibitor 3, Myeloid differentiation pri themselves detectable (e.g., fluorescent moieties, electro mary response protein MyD88, Netrin-1, Netrin-4, Neuronal chemical labels, ecl (electrochemical luminescence) labels, cell adhesion molecule, NF-kappa-B inhibitor alpha, metal chelates, colloidal metal particles, etc.) as well as mol Nidogen-1, Osteocalcin, Pappalysin-1, Poly ADP-ribose ecules that may be indirectly detected by production of a polymerase 1 (cleaved), Probetacellulin, Prostate-specific detectable reaction product (e.g., enzymes such as horserad antigen, Prostatic acid phosphatase, Protein NOV homolog, ish peroxidase, alkaline phosphatase, etc.) or through the use Protransforming growth factor alpha, P-selectinglycoprotein of a specific binding molecule which itself may be detectable ligand 1, Sex hormone-binding globulin, SL cytokine, Tena (e.g., a labeled antibody that binds to the second antibody, Scin, Thrombospondin-2, Thymic stromal lymphopoietin, biotin, digoxigenin, maltose, oligohistidine, 2,4-dintroben Transmembrane glycoprotein NMB, Trefoil factor 3, Tubu Zene, phenylarsenate, ssDNA, dsDNA, etc.). lointerstitial nephritis antigen, Tumor necrosis factor receptor 0032 Generation of a signal from the signal development superfamily member 10B, Tumor necrosis factor receptor element can be performed using various optical, acoustical, superfamily member 8, Versican core protein, Bone morpho and electrochemical methods well known in the art. genetic protein 7, Carbonic anhydrase 9, Caspase-9, Colla Examples of detection modes include fluorescence, radio genase 3, Granzyme M. Heparin-binding EGF-like growth chemical detection, reflectance, absorbance, amperometry, factor, Insulin receptor Substrate 1, Keratin, type II cytoskel conductance, impedance, interferometry, ellipsometry, etc. In etal 6 (6A, -6B, -6C mix), Myeloid differentiation primary certain of these methods, the solid phase antibody is coupled response protein MyD88, SL cytokine, Vascular endothelial to a transducer (e.g., a diffraction grating, electrochemical growth factor D. Vascular endothelial growth factor receptor sensor, etc) for generation of a signal, while in others, a signal 2, and Vascular endothelial growth factor receptor 3 or one or is generated by a transducer that is spatially separate from the more markers related thereto, are correlated to the status of Solid phase antibody (e.g., a fluorometer that employs an the sepsis patient. As described herein, measurement of one excitation light source and an optical detector). This list is not or more sepsis biomarkers of the present invention may be meant to be limiting. Antibody-based biosensors may also be used, individually or in panels comprising a plurality of sepsis employed to determine the presence or amount of analytes biomarkers, for identifying a subject suffering from SIRS, that optionally eliminate the need for a labeled molecule. sepsis, severe sepsis, septic shock and/or MODS, for distin US 2015/0293131 A1 Oct. 15, 2015

guishing amongst these conditions, or for assigning a prog nine of greater than or equal to 50% (1.5-fold from baseline), nosis to a subject Suffering from one or more of these condi or a reduction in urine output (documented oliguria of less tions, etc. than 0.5 ml/kg per hour for at least 6 hours). This term is 0034) For purposes of this document, the following defi synonymous with “acute kidney injury' or “AKI.” nitions apply: 0043. As used herein, the term “relating a signal to the 0035. As used herein, “SIRS refers to a condition that presence or amount of an analyte reflects the following exhibits two or more of the following: understanding. Assay signals are typically related to the pres a temperature >38°C. or <36° C.: ence or amount of an analyte through the use of a standard a heart rate of >90 beats per minute (tachycardia); curve calculated using known concentrations of the analyte of a respiratory rate of >20 breaths per minute (tachypnea) or a interest. As the term is used herein, an assay is “configured to PCO<4.3 kPa; and detect an analyte if an assay can generate a detectable signal a white blood cell count >12,000 per mm, <4,000 per mm. indicative of the presence or amount of a physiologically or >10% immature (band) forms. relevant concentration of the analyte. Because an antibody 0036) As used herein, “Sepsis” refers to SIRS, further epitope is on the order of 8 amino acids, an immunoassay accompanied by a clinically evident or microbiologically configured to detect a marker of interest will also detect confirmed infection. This infection may be bacterial, fungal, polypeptides related to the marker sequence, so long as those parasitic, or viral. polypeptides contain the epitope(s) necessary to bind to the 0037. As used herein, “Severe sepsis” refers to a subset of antibody or antibodies used in the assay. The term “related sepsis patients, in which sepsis is further accompanied by marker” as used herein with regard to a biomarker Such as one organ hypoperfusion made evident by at least one sign of of the sepsis biomarkers described herein refers to one or organ dysfunction Such as hypoxemia, oliguria, metabolic more fragments, variants, etc., of a particular marker or its acidosis, or altered cerebral function. biosynthetic parent that may be detected as a Surrogate for the 0038. As used herein, “Septic shock” refers to a subset of marker itself or as independent biomarkers. The term also severe sepsis patients, in which severe sepsis is further refers to one or more polypeptides present in a biological accompanied by hypotension, made evident by a systolic sample that are derived from the biomarker precursor com blood pressure <90 mm Hg, or the requirement for pharma plexed to additional species, such as binding , recep ceutical intervention to maintain blood pressure. tors, heparin, lipids, Sugars, etc. 0039. As used herein, MODS (multiple organ dysfunction 0044. In this regard, the skilled artisan will understand that syndrome) is the presence of altered organ function in a the signals obtained from an immunoassay are a direct result patient who is acutely ill such that homeostasis cannot be of complexes formed between one or more antibodies and the maintained without intervention. Primary MODS is the direct target biomolecule (i.e., the analyte) and polypeptides con result of a well-defined insult in which organ dysfunction taining the necessary epitope(s) to which the antibodies bind. occurs early and can be directly attributable to the insult itself. While such assays may detect the full length biomarker and Secondary MODS develops as a consequence of a host the assay result be expressed as a concentration of a biomar response and is identified within the context of SIRS. ker of interest, the signal from the assay is actually a result of 0040. As used herein, an “injury to renal function' is an all such “immunoreactive' polypeptides present in the abrupt (within 14 days, preferably within 7 days, more pref sample. Expression of biomarkers may also be determined by erably within 72 hours, and still more preferably within 48 means other than immunoassays, including protein measure hours) measurable reduction in a measure of renal function. ments (such as dot blots, western blots, chromatographic Such an injury may be identified, for example, by a decrease methods, mass spectrometry, etc.) and nucleic acid measure in glomerular filtration rate or estimated GFR, a reduction in ments (mRNA quatitation). This list is not meant to be limit urine output, an increase in serum creatinine, an increase in ing. With regard to biomarkers which exist in one form as serum cystatin C, a requirement for renal replacement type-I, type-II, or GPI-anchored membrane proteins, such therapy, etc. “Improvement in Renal Function' is an abrupt membrane proteins typically comprise a Substantial extracel (within 14 days, preferably within 7 days, more preferably lular domain, some or all of which can be detected as soluble within 72 hours, and still more preferably within 48 hours) forms present in aqueous samples such as blood, serum, measurable increase in a measure of renal function. Preferred plasma, urine, etc., either as cleavage products or as splice methods for measuring and/or estimating GFR are described variants which delete an effective membrane spanning hereinafter. domain. Preferred assays detect soluble forms of these biom 0041. As used herein, “reduced renal function' is an arkers. abrupt (within 14 days, preferably within 7 days, more pref 0045. The term “positive going marker as that term is erably within 72 hours, and still more preferably within 48 used herein refer to a marker that is determined to be elevated hours) reduction in kidney function identified by an absolute in sepsis patients Suffering from a disease or condition, rela increase in serum creatinine of greater than or equal to 0.1 tive to sepsis patients not suffering from that disease or con mg/dL (>8.8 umol/L), a percentage increase in serum creati dition. The term “negative going marker as that term is used nine of greater than or equal to 20% (1.2-fold from baseline), herein refer to a marker that is determined to be reduced in or a reduction in urine output (documented oliguria of less sepsis patients Suffering from a disease or condition, relative than 0.5 ml/kg per hour). to sepsis patients not suffering from that disease or condition. 0042. As used herein, “acute renal failure' or ARF is an 0046. The term “subject' as used herein refers to a human abrupt (within 14 days, preferably within 7 days, more pref or non-human organism. Thus, the methods and composi erably within 72 hours, and still more preferably within 48 tions described herein are applicable to both human and vet hours) reduction in kidney function identified by an absolute erinary disease. Further, while a subject is preferably a living increase in serum creatinine of greater than or equal to 0.3 organism, the invention described herein may be used in mg/dl (>26.4 umol/l), a percentage increase in serum creati post-mortem analysis as well. Preferred Subjects are humans, US 2015/0293131 A1 Oct. 15, 2015

and most preferably “patients.” which as used herein refers to -continued living humans that are receiving medical care for a disease or condition. This includes persons with no defined illness who SwissProtNum Preferred Name are being investigated for signs of pathology. A 'sepsis P22223 Cadherin-3 patient' is a patient Suffering from sepsis. Cancer Antigen 15-3 0047 Preferably, an analyte such as a sepsis biomarker is Cancer Antigen 19-9 measured in a sample. Such a sample may be obtained from a Q16790 Carbonic anhydrase 9 P13688 Carcinoembryonic antigen-related cell adhesion patient, such as a sepsis patient. Preferred samples are body molecule 1 fluid samples. PO6731 Carcinoembryonic antigen-related cell adhesion 0048. The term “body fluid sample' as used herein refers molecule 5 to a sample of bodily fluid obtained for the purpose of diag P42574 Caspase-3, active P25774 Cathepsin S nosis, prognosis, classification or evaluation of a sepsis P22362 C-C motif chemokine 1 patient of interest, Such as a patient or transplant donor. In Q16663 C-C motif chemokine 15 certain embodiments, such a sample may be obtained for the Q92583 C-C motif chemokine 17 purpose of determining the outcome of an ongoing condition P55774 C-C motif chemokine 18 P78556 C-C motif chemokine 20 or the effect of a treatment regimen on a condition. Preferred OOO585 C-C motif chemokine 21 body fluid samples include blood, serum, plasma, cerebrospi OOO175 C-C motif chemokine 24 nal fluid, urine, saliva, Sputum, and pleural effusions. In addi P10909 Clusterin tion, one of skill in the art would realize that certain body fluid P454.52 Collagenase 3 PO1308 C-peptide (Insulin) samples would be more readily analyzed following a frac O14625 C—X-C motif chemokine 11 tionation or purification procedure, for example, separation Q9H2A7 C—X-C motif chemokine 16 of whole blood into serum or plasma components. P38936 Cyclin-dependent kinase inhibitor 1 0049. The term “diagnosis' as used herein refers to meth Q96HY6 DDRGK domain-containing protein 1 P39060 Endostatin ods by which the skilled artisan can estimate and/or deter POO533 Epidermal growth factor receptor mine the probability (“a likelihood') of whether or not a O14944 Epiregulin patient is Suffering from a given disease or condition. In the P16422 Epithelial cell adhesion molecule case of the present invention, "diagnosis' includes using the P19235 Erythropoietin receptor O95750 Fibroblast growth factor 19 results of an assay, most preferably an immunoassay, for a Q9GZV9 Fibroblast growth factor 23 sepsis biomarker of the present invention, optionally together P17931 Galectin-3 with other clinical characteristics, to arrive at a diagnosis (that PO9681 Gastric inhibitory polypeptide is, the occurrence or nonoccurrence) of a disease or condition. PO1275 Glucagon PO1275 Glucagon-like peptide 1 That such a diagnosis is “determined' is not meant to imply PO9211 Glutathione S-transferase P that the diagnosis is 100% accurate. Many biomarkers are PO4792 Heat shock protein beta-1 indicative of multiple conditions. The skilled clinician does PO5230 Heparin-binding growth factor 1 not use biomarker results in an informational vacuum, but Q96D42 Hepatitis Avims cellular receptor 1 PO8581 Hepatocyte growth factor receptor rather test results are used together with other clinical indicia PO1308 insulin to arrive at a diagnosis. Thus, a measured biomarker level on P08069 insulin-like growth factor 1 receptor one side of a predetermined diagnostic threshold indicates a P24592 insulin-like growth factor-binding protein 6 greater likelihood of the occurrence of disease in the sepsis Q16270 insulin-like growth factor-binding protein 7 PO1563 interferon alpha-2 patient relative to a measured level on the other side of the Q9NYY1 interleukin-20 predetermined diagnostic threshold. Q9HBE4 interleukin-21 0050. Similarly, a prognostic risk signals a probability (“a Q8IZJO interleukin-28A Q8IU54 interleukin-29 likelihood') that a given course or outcome will occur. A level O95760 interleukin-33 or a change in level of a prognostic indicator, which in turn is PO7476 nvolucrin associated with an increased probability of morbidity (e.g., P10997 slet amyloid polypeptide worsening sepsis or death) is referred to as being “indicative PO4264; P13645 Keratin, type II cytoskeletal 1 (Keratin-1, -10 mix) ofan increased likelihood' of an adverse outcome inapatient. Lymphatic vessel endothelial hyaluronic acid 0051 Sepsis Biomarkers receptor 1 0052. The following table provides a list of the sepsis P39900 Macrophage metalloelastase biomarkers of the present invention, together with the Swiss P14780; P16035 Matrix metalloproteinase-9: Metalloproteinase complex inhibitor 2 Prot entry number for the human precursor. P35625 Metalloproteinase inhibitor 3 Q99836 Myeloid differentiation primary response protein MyD88 SwissProtNum Preferred Name O95631 Netrin-1 Q9HB63 Netrin-4 PO1023 Alpha-2 macroglobulin Neuronal cell adhesion molecule PO3950 Angiogenin Q92823 Q02763 Angiopoietin-1 receptor P25963 NF-kappa-B inhibitor alpha Q9Y5C1 Angiopoietin-related protein 3 P14543 Nidogen-1 Q9BY76 Angiopoietin-related protein 4 PO2818 Osteocalcin Q8NI99 Angiopoietin-related protein 6 Q13219 Pappalysin-1 P02652 Apollipoprotein A-II PO9874 Poly ADP-ribose polymerase 1 (cleaved) PO2656 Apollipoprotein C-III P35070 Probetacellulin PO8519 Apollipoprotein(a) PO7288 Prostate-specific antigen P18075 Bone morphogenetic protein 7 P15309 Prostatic acid phosphatase O75309 Cadherin-16 P48745 Protein NOV homolog US 2015/0293131 A1 Oct. 15, 2015

-continued immunoassays (ELISA), radioimmunoassays (RIAS), com petitive binding assays, and the like. SwissProtNum Preferred Name 0056 Antibodies or other polypeptides may be immobi PO1135 Protransforming growth factor alpha lized onto a variety of solid Supports for use in assays. Solid Q14242 P-selectinglycoprotein ligand 1 phases that may be used to immobilize specific binding mem PO4278 Sex hormone-binding globulin bers include those developed and/or used as Solid phases in P49711 SL cytokine Solid phase binding assays. Examples of suitable Solid phases P24821 Tenascin P3S442 Thrombospondin-2 include membrane filters, cellulose-based papers, beads (in Q969D9 Thymic stromal lymphopoietin cluding polymeric, latex and paramagnetic particles), glass, Q14956 Transmembrane glycoprotein NMB silicon wafers, microparticles, nanoparticles, TentaGels, Q07654 Trefoil factor 3 AgroGels, PEGA gels, SPOCC gels, and multiple-well Q9UJW2 Tubulointerstitial nephritis antigen O14763 Tumor necrosis factor receptor Superfamily plates. An assay strip could be prepared by coating the anti member 10B body or a plurality of antibodies in an array on Solid Support. P28908 Tumor necrosis factor receptor Superfamily This strip could then be dipped into the test sample and then member 8 processed quickly through washes and detection steps togen P13611 Versican core protein P18075 Bone morphogenetic protein 7 erate a measurable signal. Such as a colored spot. Antibodies Q16790 Carbonic anhydrase 9 or other polypeptides may be bound to specific Zones of assay P55211 Caspase-9 devices either by conjugating directly to an assay device P454.52 Collagenase 3 PS1124 Granzyme M Surface, or by indirect binding. In an example of the later case, Q99075 Heparin-binding EGF-like growth factor antibodies or other polypeptides may be immobilized on par P35568 Insulin receptor substrate 1 ticles or other Solid Supports, and that Solid Support immobi PO2538; PO4259; P4.8668 Keratin, type II cytoskeletal 6 (6A, -6B, -6C lized to the device surface. mix) Q99836 Myeloid differentiation primary response 0057 Biological assays require methods for detection, protein MyD88 and one of the most common methods for quantitation of P49771 SL cytokine results is to conjugate a detectable label to a protein or nucleic O43915 Vascular endothelial growth factor D acid that has affinity for one of the components in the biologi P35968 Vascular endothelial growth factor receptor 2 cal system being studied. Detectable labels may include mol P35916 Vascular endothelial growth factor receptor 3 ecules that are themselves detectable (e.g., fluorescent moi eties, electrochemical labels, metal chelates, etc.) as well as 0053 Marker Assays molecules that may be indirectly detected by production of a 0054. In general, immunoassays involve contacting a detectable reaction product (e.g., enzymes Such as horserad sample containing or Suspected of containing a biomarker of ish peroxidase, alkaline phosphatase, etc.) or by a specific interest with at least one antibody that specifically binds to the binding molecule which itself may be detectable (e.g., biotin, biomarker. A signal is then generated indicative of the pres digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, ence or amount of complexes formed by the binding of phenylarsenate, ssDNA, dsDNA, etc.). polypeptides in the sample to the antibody. The signal is then 0.058 Preparation of solid phases and detectable label con related to the presence or amount of the biomarker in the jugates often comprise the use of chemical cross-linkers. sample. Numerous methods and devices are well known to Cross-linking reagents contain at least two reactive groups, the skilled artisan for the detection and analysis of biomark and are divided generally into homofunctional cross-linkers ers. See, e.g., U.S. Pat. Nos. 6,143,576; 6,113,855; 6,019,944; (containing identical reactive groups) and heterofunctional 5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; cross-linkers (containing non-identical reactive groups). 5,851,776; 5,824,799; 5,679,526; 5,525,524; and 5,480,792, Homobifunctional cross-linkers that couple through amines, and The Immunoassay Handbook, David Wild, ed. Stockton Sulfhydryls or react non-specifically are available from many Press, New York, 1994, each of which is hereby incorporated commercial sources. Maleimides, alkyl and aryl halides, by reference in its entirety, including all tables, figures and alpha-haloacyls and pyridyl disulfides are thiol reactive claims. groups. Maleimides, alkyl and aryl halides, and alpha-haloa 0055. The assay devices and methods known in the art can cyls react with sulfhydryls to form thiol ether bonds, while utilize labeled molecules in various sandwich, competitive, or pyridyl disulfides react with sulfhydryls to produce mixed non-competitive assay formats, to generate a signal that is disulfides. The pyridyl disulfide product is cleavable Imi related to the presence or amount of the biomarker of interest. doesters are also very useful for protein-protein cross-links. A Suitable assay formats also include chromatographic, mass variety of heterobifunctional cross-linkers, each combining spectrographic, and protein “blotting methods. Additionally, different attributes for Successful conjugation, are commer certain methods and devices, such as biosensors and optical cially available. immunoassays, may be employed to determine the presence 0059. In certain aspects, the present invention provides or amount of analytes without the need for a labeled mol kits for the analysis of the described sepsis biomarkers. The ecule. See, e.g., U.S. Pat. Nos. 5,631,171; and 5,955.377, kit comprises reagents for the analysis of at least one test each of which is hereby incorporated by reference in its sample which comprise at least one antibody that binds a entirety, including all tables, figures and claims. One skilled sepsis biomarker. The kit can also include devices and in the art also recognizes that robotic instrumentation includ instructions for performing one or more of the diagnostic ing but not limited to Beckman ACCESSR, Abbott and/or prognostic correlations described herein. Preferred AXSYMR), Roche ELECSYS(R), Dade Behring STRATUS(R) kits will comprise an antibody pair for performing a sandwich systems are among the immunoassay analyzers that are assay, or a labeled species for performing a competitive assay, capable of performing immunoassays. But any Suitable for the analyte. Preferably, an antibody pair comprises a first immunoassay may be utilized, for example, enzyme-linked antibody conjugated to a solid phase and a second antibody US 2015/0293131 A1 Oct. 15, 2015

conjugated to a detectable label, wherein each of the first and well known in the art. See, e.g., van Erp et al., J. Immunoassay second antibodies bind a sepsis biomarker. Most preferably 12: 425-43, 1991; Nelson and Griswold, Comput. Methods each of the antibodies are monoclonal antibodies. The Programs Biomed. 27: 65-8, 1988. instructions for use of the kit and performing the correlations 0064. The term “epitope” refers to an antigenic determi can be in the form of labeling, which refers to any written or nant capable of specific binding to an antibody. Epitopes recorded material that is attached to, or otherwise accompa usually consist of chemically active surface groupings of nies a kit at any time during its manufacture, transport, sale or molecules Such as amino acids or Sugar side chains and usu use. For example, the term labeling encompasses advertising ally have specific three dimensional structural characteristics, leaflets and brochures, packaging materials, instructions, as well as specific charge characteristics. Conformational and audio or video cassettes, computer discs, as well as writing nonconformational epitopes are distinguished in that the imprinted directly on kits. binding to the former but not the latter is lost in the presence 0060 Antibodies of denaturing solvents. 0061 The term “antibody” as used herein refers to a pep 0065. Numerous publications discuss the use of phage tide or polypeptide derived from, modeled after or substan display technology to produce and screen libraries of tially encoded by an immunoglobulin or immunoglobu polypeptides for binding to a selected analyte. See, e.g., lin , or fragments thereof, capable of specifically Cwirla et al., Proc. Natl. Acad. Sci. USA 87, 6378-82, 1990; binding an antigen or epitope. See, e.g. Fundamental Immu Devlin et al., Science 249, 404-6, 1990, Scott and Smith, nology, 3rd Edition, W. E. Paul, ed., Raven Press, N.Y. Science 249, 386-88, 1990; and Ladner et al., U.S. Pat. No. (1993); Wilson (1994; J. Immunol. Methods 175:267-273: 5,571,698. A basic concept of phage display methods is the Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97. establishment of a physical association between DNA encod The term antibody includes antigen-binding portions, i.e., ing a polypeptide to be screened and the polypeptide. This 'antigen binding sites.” (e.g., fragments, Subsequences, physical association is provided by the phage particle, which complementarity determining regions (CDRS)) that retain displays a polypeptide as part of a capsid enclosing the phage capacity to bind antigen, including (i) a Fab fragment, a genome which encodes the polypeptide. The establishment of monovalent fragment consisting of the VL, VH, CL and CH1 a physical association between polypeptides and their genetic domains; (ii) a F(ab')2 fragment, a bivalent fragment com material allows simultaneous mass screening of very large prising two Fab fragments linked by a disulfide bridge at the numbers of phage bearing different polypeptides. Phage dis hinge region; (iii) a Fd fragment consisting of the VHand CH1 playing a polypeptide with affinity to a target bind to the target domains; (iv) a Fv fragment consisting of the VL and VH and these phage are enriched by affinity screening to the domains of a single arm of an antibody, (v) a dAb fragment target. The identity of polypeptides displayed from these (Wardet al., (1989) Nature 341:544-546), which consists of a phage can be determined from their respective genomes. VH domain; and (vi) an isolated complementarity determin Using these methods a polypeptide identified as having a ing region (CDR). Single chain antibodies are also included binding affinity for a desired target can then be synthesized in by reference in the term “antibody.” bulk by conventional means. See, e.g., U.S. Pat. No. 6,057. 0062 Antibodies used in the immunoassays described 098, which is hereby incorporated in its entirety, including all herein preferably specifically bind to a sepsis biomarker of tables, figures, and claims. the present invention. The term “specifically binds” is not 0066. The antibodies that are generated by these methods intended to indicate that an antibody binds exclusively to its may then be selected by first screening for affinity and speci intended target since, as noted above, an antibody binds to any ficity with the purified polypeptide of interestand, ifrequired, polypeptide displaying the epitope(s) to which the antibody comparing the results to the affinity and specificity of the binds. Rather, an antibody “specifically binds' if its affinity antibodies with polypeptides that are desired to be excluded for its intended target is about 5-fold greater when compared from binding. The screening procedure can involve immobi to its affinity for a non-target molecule which does not display lization of the purified polypeptides in separate wells of the appropriate epitope(s). Preferably the affinity of the anti microtiterplates. The Solution containing a potential antibody body will be at least about 5 fold, preferably 10 fold, more or groups of antibodies is then placed into the respective preferably 25-fold, even more preferably 50-fold, and most microtiter wells and incubated for about 30 minto 2 h. The preferably 100-fold or more, greater for a target molecule microtiter wells are then washed and a labeled secondary than its affinity for a non-target molecule. In preferred antibody (for example, an anti-mouse antibody conjugated to embodiments, Preferred antibodies bind with affinities of at alkaline phosphatase if the raised antibodies are mouse anti least about 107M, and preferably between about 10 M' to bodies) is added to the wells and incubated for about 30 min about 10M, about 10M to about 10'M' or about 10' and then washed. Substrate is added to the wells and a color M' to about 10* M. reaction will appear where antibody to the immobilized I0063 Affinity is calculated as K. k/k (kis the dis polypeptide(s) are present. Sociation rate constant, K is the association rate constant 0067. The antibodies so identified may then be further and K is the equilibrium constant). Affinity can be deter analyzed for affinity and specificity in the assay design mined at equilibrium by measuring the fraction bound (r) of selected. In the development of immunoassays for a target labeled ligand at various concentrations (c). The data are protein, the purified target protein acts as a standard with graphed using the Scatchard equation: ric-K(n-r): where which to judge the sensitivity and specificity of the immu r-moles of bound ligand/mole of receptor at equilibrium; noassay using the antibodies that have been selected. Because c-free ligand concentration at equilibrium; K equilibrium the binding affinity of various antibodies may differ; certain association constant; and n number of ligand binding sites antibody pairs (e.g., in Sandwich assays) may interfere with per receptor molecule. By graphical analysis, r/c is plotted on one another sterically, etc., assay performance of an antibody the Y-axis versus r on the X-axis, thus producing a Scatchard may be a more important measure than absolute affinity and plot. Antibody affinity measurement by Scatchard analysis is specificity of an antibody. US 2015/0293131 A1 Oct. 15, 2015

0068 While the present application describes antibody 0074. In this context, “diseased' is meant to refer to a based binding assays in detail, alternatives to antibodies as population having one characteristic (the presence of a dis binding species in assays are well known in the art. These ease or condition or the occurrence of some outcome) and include receptors for a particular target, aptamers, etc. “nondiseased' is meant to refer to a population lacking the Aptamers are oligonucleic acid or peptide molecules that characteristic. While a single decision threshold is the sim bind to a specific target molecule. Aptamers are usually cre plest application of such a method, multiple decision thresh ated by selecting them from a large random sequence pool, olds may be used. For example, below a first threshold, the but natural aptamers also exist. High-affinity aptamers con absence of disease may be assigned with relatively high con taining modified nucleotides can confer improved character fidence, and above a second threshold the presence of disease istics on the ligand. Such as improved in Vivo stability or may also be assigned with relatively high confidence. improved delivery characteristics. Examples of such modifi Between the two thresholds may be considered indetermi cations include chemical Substitutions at the ribose and/or nate. This is meant to be exemplary in nature only. phosphate and/or base positions, and may includeamino acid 0075. In addition to threshold comparisons, other methods side chain functionalities. for correlating assay results to a patient classification (occur 0069. Assay Correlations rence or nonoccurrence of disease, likelihood of an outcome, 0070 The term “correlating as used herein in reference to etc.) include decision trees, rule sets, Bayesian methods, and the use of biomarkers refers to comparing the presence or neural network methods. These methods can produce prob amount of the biomarker(s) in a patient to its presence or ability values representing the degree to which a sepsis amount in persons known to suffer from, or known to be at patient belongs to one classification out of a plurality of risk of a given condition; or in persons known to be free of a classifications. given condition. Often, this takes the form of comparing an 0076 Measures of test accuracy may be obtained as assay result in the form of a biomarker concentration to a described in Fischer et al., Intensive Care Med. 29: 1043-51, predetermined threshold selected to be indicative of the 2003, and used to determine the effectiveness of a given occurrence or nonoccurrence of a disease or the likelihood of biomarker. These measures include sensitivity and specific Some future outcome. ity, predictive values, likelihood ratios, diagnostic odds 0071. Selecting a diagnostic threshold involves, among ratios, and ROC curve areas. The area under the curve other things, consideration of the probability of disease, dis (“AUC”) of a ROC plot is equal to the probability that a tribution of true and false diagnoses at different test thresh classifier will rank a randomly chosen positive instance olds, and estimates of the consequences of treatment (or a higher than a randomly chosen negative one. The area under failure to treat) based on the diagnosis. For example, when the ROC curve may be thought of as equivalent to the Mann considering administering a specific therapy which is highly Whitney U test, which tests for the median difference efficacious and has a low level of risk, few tests are needed between scores obtained in the two groups considered if the because clinicians can accept Substantial diagnostic uncer groups are of continuous data, or to the Wilcoxon test of tainty. On the other hand, in situations where treatment ranks. options are less effective and more risky, clinicians often need 0077. As discussed above, suitable tests may exhibit one a higher degree of diagnostic certainty. Thus, cost/benefit or more of the following results on these various measures: a analysis is involved in selecting a diagnostic threshold. specificity of greater than 0.5, preferably at least 0.6, more 0072 Suitable thresholds may be determined in a variety preferably at least 0.7, still more preferably at least 0.8, even of ways. For example, one recommended diagnostic thresh more preferably at least 0.9 and most preferably at least 0.95, old for the diagnosis of acute myocardial infarction using with a corresponding sensitivity greater than 0.2, preferably cardiac troponin is the 97.5th percentile of the concentration greater than 0.3, more preferably greater than 0.4, still more seen in a normal population. Another method may be to look preferably at least 0.5, even more preferably 0.6, yet more at serial samples from the same patient, where a prior “base preferably greater than 0.7, still more preferably greater than line result is used to monitor for temporal changes in a 0.8, more preferably greater than 0.9, and most preferably biomarker level. greater than 0.95; a sensitivity of greater than 0.5, preferably 0073 Population studies may also be used to select a deci at least 0.6, more preferably at least 0.7, still more preferably sion threshold. Receiver Operating Characteristic (“ROC) at least 0.8, even more preferably at least 0.9 and most pref arose from the field of signal detection theory developed erably at least 0.95, with a corresponding specificity greater during World War II for the analysis of radar images, and than 0.2, preferably greater than 0.3, more preferably greater ROC analysis is often used to select a threshold able to best than 0.4, still more preferably at least 0.5, even more prefer distinguish a “diseased subpopulation from a “nondiseased ably 0.6, yet more preferably greater than 0.7, still more Subpopulation. A false positive in this case occurs when the preferably greater than 0.8, more preferably greater than 0.9, person tests positive, but actually does not have the disease. A and most preferably greater than 0.95; at least 75% sensitiv false negative, on the other hand, occurs when the person tests ity, combined with at least 75% specificity; a ROC curve area negative, Suggesting they are healthy, when they actually do of greater than 0.5, preferably at least 0.6, more preferably have the disease. To draw a ROC curve, the true positive rate 0.7., still more preferably at least 0.8, even more preferably at (TPR) and false positive rate (FPR) are determined as the least 0.9, and most preferably at least 0.95; an odds ratio decision threshold is varied continuously. Since TPR is different from 1, preferably at least about 2 or more or about equivalent with sensitivity and FPR is equal to 1-specificity, 0.5 or less, more preferably at least about 3 or more or about the ROC graph is sometimes called the sensitivity vs 0.33 or less, still more preferably at least about 4 or more or (1-specificity) plot. A perfect test will have an area under the about 0.25 or less, even more preferably at least about 5 or ROC curve of 1.0; a random test will have an area of 0.5. A more or about 0.2 or less, and most preferably at least about threshold is selected to provide an acceptable level of speci 10 or more or about 0.1 or less; a positive likelihood ratio ficity and sensitivity. (calculated as sensitivity/(1-specificity)) of greater than 1, at US 2015/0293131 A1 Oct. 15, 2015

least 2, more preferably at least 3, still more preferably at least Inclusion Criteria 5, and most preferably at least 10; and or a negative likelihood I008.4 males and females 18 years of age or older; ratio (calculated as (1-sensitivity)/specificity) of less than 1, Study population 1: approximately 300 patients that have at less than or equal to 0.5, more preferably less than or equal to least one of: 0.3, and most preferably less than or equal to 0.1 shock (SBP <90 mmHg and/or need for vasopressor support to maintain MAPD60 mmHg and/or documented drop in SBP 0078. Additional clinical indicia may be combined with of at least 40 mmHg); and the sepsis biomarker assay result(s) of the present invention. sepsis: Other clinical indicia which may be combined with the sepsis Study population 2: approximately 300 patients that have at biomarker assay result(s) of the present invention includes least one of: demographic information (e.g., weight, sex, age, race), medi IV antibiotics ordered in computerized physician order entry cal history (e.g., family history, type of Surgery, pre-existing (CPOE) within 24 hours of enrollment: disease Such as aneurism, congestive heart failure, preec contrast media exposure within 24 hours of enrollment; lampsia, eclampsia, diabetes mellitus, hypertension, coro increased Intra-Abdominal Pressure with acute decompen nary artery disease, proteinuria, or renal insufficiency), risk sated heart failure; and severe trauma as the primary reason scores (APACHE score, PREDICT score, TIMI Risk Score for ICU admission and likely to be hospitalized in the ICU for 48 hours after enrollment; for UA/NSTEMI, Framingham Risk Score), a urine total Study population 3: approximately 300 patients expected to protein measurement, a glomerular filtration rate, an esti be hospitalized through acute care setting (ICU or ED) with a mated glomerular filtration rate, a urine production rate, a known risk factor for acute renal injury (e.g. sepsis, hypoten serum or plasma creatinine concentration, a renal papillary sion/shock (Shock systolic BP<90 mmHg and/or the need antigen 1 (RPA1) measurement; a renal papillary antigen 2 for vasopressor support to maintain a MAPD60 mmHg and/or (RPA2) measurement; a urine creatinine concentration, a a documented drop in SBP>40 mmHg), major trauma, hem fractional excretion of sodium, aurine sodium concentration, orrhage, or major Surgery); and/or expected to be hospitalized a urine creatinine to serum or plasma creatinine ratio, a urine to the ICU for at least 24 hours after enrollment; specific gravity, a urine osmolality, a urine urea nitrogen to Study population 4: approximately 1000 patients that are 21 plasma urea nitrogen ratio, a plasma BUN to creatnine ratio, years of age or older, within 24 hours of being admitted into and/or a renal failure index calculated as urine sodium/(urine the ICU, expected to have an indwelling urinary catheter for creatinine/plasma creatinine). at least 48 hours after enrollment, and have at least one of the 0079 Combining assay results/clinical indicia in this following acute conditions within 24 hours prior to enroll manner can comprise the use of multivariate logistical regres ment: Sion, loglinear modeling, neural network analysis, n-of-m (i) respiratory SOFA score of >2 (PaO2/FiO2<300), (ii) car analysis, decision tree analysis, etc. This list is not meant to be diovascular SOFA score of >1 (MAP <70 mm Hg and/or any limiting. vasopressor required). 0080 Selecting a Treatment Regimen Exclusion Criteria I0085 known pregnancy; 0081. Once a diagnosis is obtained, the clinician can institutionalized individuals; readily select a treatment regimen that is compatible with the previous renal transplantation; diagnosis. The skilled artisan is aware of appropriate treat known acutely worsening renal function prior to enrollment ments for numerous diseases discussed in relation to the (e.g., any category of RIFLE criteria); methods of diagnosis described herein. See, e.g., Merck received dialysis (either acute or chronic) within 5 days prior Manual of Diagnosis and Therapy, 17th Ed. Merck Research to enrollment or in imminent need of dialysis at the time of Laboratories, Whitehouse Station, N.J., 1999. In addition, enrollment; since the methods and compositions described herein provide known infection with human immunodeficiency virus (HIV) prognostic information, the markers of the present invention or a hepatitis virus; may be used to monitor a course of treatment. For example, meets any of the following: improved or worsened prognostic state may indicate that a (i) active bleeding with an anticipated need for >4 units PRBC particular treatment is or is not efficacious. in a day; 0082 One skilled in the art readily appreciates that the (ii) hemoglobin <7 g/dL: present invention is well adapted to carry out the objects and (iii) any other condition that in the physician's opinion would obtain the ends and advantages mentioned, as well as those contraindicate drawing serial blood samples for clinical study inherent therein. The examples provided herein are represen purposes; tative of preferred embodiments, are exemplary, and are not meets only the SBP <90 mmHg inclusion criterion set forth intended as limitations on the scope of the invention. above, and does not have shock in the attending physicians or principal investigator's opinion; I0086. After obtaining informed consent, an EDTA anti Example 1 coagulated blood sample (10 mL) and a urine sample (25-50 mL) are collected from each patient. Blood and urine samples Sepsis Patient Sample Collection are then collected at 4 (+0.5) and 8 (+1) hours after contrast administration (if applicable); at 12 (+1), 24 (+2), 36 (+2), 48 0083. The objective of this study is to collect samples from (+2), 60 (+2), 72 (+2), and 84 (+2) hours after enrollment, and acutely ill patients. Approximately 1900 adults expected to be thereafter daily up to day 7 to day 14 while the subject is in the ICU for at least 48 hours will be enrolled. To be enrolled hospitalized. Blood is collected via direct venipuncture or via in the study, each patient must meet all of the following other available venous access. Such as an existing femoral inclusion criteria and none of the following exclusion criteria: sheath, central venous line, peripheral intravenous line or US 2015/0293131 A1 Oct. 15, 2015 hep-lock. These study blood samples are processed to plasma and analyses using the measured analyte concentrations were at the clinical site, frozen and shipped to Astute Medical, Inc., the same as in Example 3 above. San Diego, Calif. The study urine samples are frozen and shipped to Astute Medical, Inc. Example 5 Example 2 Use of Analyte as a Marker for Septic Shock Immunoassay Format 0091. Two cohorts were defined as (Cohort 1) patients who had both sepsis and hypotension on the same day (septic 0087 Analytes are measured using standard sandwich shock), and (Cohort 2) patients who did not have septic shock enzyme immunoassay techniques. A first antibody which on any day from enrollment to 6 days after. A patient was binds the analyte is immobilized in wells of a 96 well poly classified as having hypotension if his/her systolic blood styrene microplate. Analyte standards and test samples are pressure was below 90 mm Hg. The urine and plasma sample pipetted into the appropriate wells and any analyte present is collections and analyses using the measured analyte concen bound by the immobilized antibody. After washing away any unbound Substances, a horseradish peroxidase-conjugated trations were the same as in Example 3 above. second antibody which binds the analyte is added to the wells, Example 6 thereby forming sandwich complexes with the analyte (if present) and the first antibody. Following a wash to remove any unbound antibody-enzyme reagent, a Substrate Solution Use of Analyte as a Marker for Progression to Septic comprising tetramethylbenzidine and hydrogen peroxide is Shock added to the wells. Color develops in proportion to the 0092. Two cohorts were defined as (Cohort 1) patients amount of analyte present in the sample. The color develop who did not have septic shock (see Example 5 for definition) ment is stopped and the intensity of the color is measured at on any of the 5 days prior to enrollment but had septic shock 540 nm or 570 nm. An analyte concentration is assigned to the on any of the Subsequent 7 days (enrollment to 6 days after), test sample by comparison to a standard curve determined and (Cohort 2) patients who did not have septic shock on any from the analyte standards. of the 5 days prior to enrollment and on any of the Subsequent 7 days. The urine and plasma sample collections and analyses Example 3 using the measured analyte concentrations were the same as in Example 3 above. Use of Analyte as a Marker for Sepsis I0088 Patients from the intensive care unit (ICU) were Example 7 classified as positive or negative for sepsis according to clini cal diagnosis on each day from enrollment to 6 days after. Use of Analyte as a Marker for Death with Sepsis 0089. Two cohorts were defined as (Cohort 1) patients who had sepsis, and (Cohort 2) patients who did not have 0093. Two cohorts were defined as (Cohort 1) patients sepsis on any day from enrollment to 6 days after (7 days who had sepsis on any day from enrollment to 6 days after and total). Both urine and plasma samples from each patient in died within 30 days after enrollment, and (Cohort 2) patients Cohorts 1 and 2 were collected on the day of enrollment. The who did not die and patients who died within 30 days after concentrations of the analyte in these samples were measured enrollment but did not have sepsis on any day from enroll by Standard immunoassay methods using commercially ment to 6 days after. The urine and plasma sample collections available assay reagents. A receiver operating characteristic and analyses using the measured analyte concentrations were (ROC) curve was generated using the concentrations, and the the same as in Example 3 above. performance of the analyte was assessed by the area under the ROC curve (AUC). The two-tailed p-value of the AUC for the Example 8 analyte was calculated, and if the p-value was less than 0.1. the AUC was considered statistically significant. Use of Analyte as a Marker for Death with Septic Shock Example 4 0094. Two cohorts were defined as (Cohort 1) patients who had septic shock (see Example 5 for definition) on any Use of Analyte as a Marker for Progression to Sepsis day from enrollment to 6 days after and died within 30 days 0090 Two cohorts were defined as (Cohort 1) patients after enrollment, and (Cohort 2) patients who did not die and who did not have sepsis during the 5 days prior to enrollment patients who died within 30 days after enrollment but did not but had sepsis on any of the Subsequent 7 days (enrollment to have septic shock on any day from enrollment to 6 days after. 6 days after), and (Cohort 2) patients who did not have sepsis The urine and plasma sample collections and analyses using on any of the 5 days prior to enrollment and on any of the the measured analyte concentrations were the same as in Subsequent 7 days. The urine and plasma sample collections Example 3 above. TABLE 1. Use of Biomarkers in Sepsis Preferred Name 1 2 3 4 S 6 7 8 9 10 11 12

Alpha-2 macroglobulin S S Angiogenin US 2015/0293131 A1 Oct. 15, 2015 13

TABLE 1-continued Use of Biomarkers in Sepsis Preferred Name 1 2 3 4 S 6 7 8 9 10 11 12 Angiopoietin-1 receptor S Angiopoietin-related protein 3 S Angiopoietin-related protein 4 S S Angiopoietin-related protein 6 Apollipoprotein A-II S S S S Apollipoprotein C-III S S S S S S Apollipoprotein(a) Cadherin-16 S S S Cadherin-3 S S S S S S Cancer Antigen 19-9 S Carcinoembryonic antigen-related cell adhesion S S S S S molecule 1 Carcinoembryonic antigen-related cell adhesion S S S S S molecule 5 Caspase-3, active S S S Cathepsin S S S S S S C-C motif chemokine 1 s s s S C-C motif chemokine 15 S S S S S S S C-C motif chemokine 17 C-C motif chemokine 18 S S S C-C motif chemokine 20 s s s C-C motif chemokine 21 S S S C-C motif chemokine 24 Clusterin S S S S C-peptide (Insulin) C-X-C motif chemokine 11 S S S S C-X-C motif chemokine 16 S S S S Cyclin-dependent kinase inhibitor 1 S S S S S S S DDRGK domain-containing protein 1 Epidermal growth factor receptor Epiregulin S Epithelial cell adhesion molecule Erythropoietin receptor s S S Fibroblast growth factor 19 S S S Fibroblast growth factor 23 S S Galectin-3 S S Gastric inhibitory polypeptide S S S s Glucagon-like peptide 1 S S (GLP-1: aa98-127; aa98-128)) Glutathione S-transferase P S Heat shock protein beta-1 S Heparin-binding growth factor 1 S Hepatocyte growth factor receptor insulin-like growth factor 1 receptor insulin-like growth factor-binding protein 6 S S S insulin-like growth factor-binding protein 7 interferon alpha-2 interleukin-20 interleukin-21 S interleukin-28A interleukin-29 s s S S S interleukin-33 S nvolucrin S S slet amyloid polypeptide S Keratin, type II cytoskeletal 1 (Keratin-1, -10 mix) S S Lymphatic vessel endothelial hyaluronic S S S S acid receptor 1 Macrophage metalloelastase S S S Matrix metalloproteinase-9:Metalloproteinase S S inhibitor 2 complex Metalloproteinase inhibitor 3 S S S S S Netrin-1 S S S S S S Netrin-4 S S s s Neuronal cell adhesion molecule S S S NF-kappa-B inhibitor alpha S S S S Nidogen-1 Osteocalcin Pappalysin-1 S S S s s s S s Poly ADP-ribose polymerase 1 (cleaved) Probetacellulin Prostate-specific antigen S Prostatic acid phosphatase S S US 2015/0293131 A1 Oct. 15, 2015

TABLE 1-continued Use of Biomarkers in Sepsis Preferred Name 1 2 3 4 5 7 8 9 10 11 12 Protein NOV homolog S Protransforming growth factor alpha S S S S S S P-selectinglycoprotein ligand 1 S S S S S S S Sex hormone-binding globulin S Tenascin S Thrombospondin-2 S S Thymic stromal lymphopoietin S S S Transmembrane glycoprotein NMB S S S Trefoil factor 3 S S S S Tubulointerstitial nephritis antigen S Tumor necrosis factor receptor Superfamily member S S S S OB Tumor necrosis factor receptor Superfamily member 8 S S Versican core protein S S S S S Key for the 12 different uses of the biomarkers in Table 1: = use of analyte in urine as a marker for sepsis 2 = use of analyte in urine as a marker for progression to sepsis 3 = use of analyte in urine as a marker for septic shock 4 = use of analyte in urine as a marker for progression to septic shock 5 = use of analyte in urine as a marker for death with sepsis 6 = use of analyte in urine as a marker for death with septic shock 7 = use of analyte in plasma as a marker for sepsis 8 = use of analyte in plasma as a marker for progression to sepsis 9 = use of analyte in plasma as a marker for septic shock 10 = use of analyte in plasma as a marker for progression to septic shock 11 = use of analyte in plasma as a marker for death with sepsis 12 = use of analyte in plasma as a marker for death with septic shock S = two-tailed p-value < 0.10 for AUC

Example 9 (ii) hemoglobin <7 g/dL: (iii) any other condition that in the physician's opinion would Sepsis Patient Sample Collection (2) contraindicate drawing serial blood samples for clinical study 0095. The objective of this study is to collect samples from purposes; acutely ill patients. Approximately 800 adults expected to be meets only the SBP <90 mmHg inclusion criterion set forth in the ICU for at least 48 hours will be enrolled. To be enrolled above, and does not have shock in the attending physicians or in the study, each patient must meet all of the following principal investigator's opinion; inclusion criteria and none of the following exclusion criteria: 0098. After obtaining informed consent, an EDTA anti coagulated blood sample (10 mL) and a urine sample (25-50 Inclusion Criteria mL) are collected from each patient. Blood and urine samples are then collected at 12 (+1), 24 (+2), 36 (+2), 48 (+2), 60 0096 males and females 21 years of age or older, within (+2), 72 (+2), and 84 (+2) hours after enrollment, and there 24 hours of being admitted into the ICU, expected to have an after daily up to day 7 to day 14 while the subject is hospital indwelling urinary catheter for at least 48 hours after enroll ized. Blood is collected via direct venipuncture or via other ment, and have at least one of the following acute conditions available venous access. Such as an existing femoral sheath, within 24 hours prior to enrollment: central venous line, peripheral intravenous line or hep-lock. (i) respiratory SOFA score of >2 (PaC2/FiO2<300), (ii) car These study blood samples are processed to plasma at the diovascular SOFA score of >1 (MAP <70 mm Hg and/or any clinical site, frozen and shipped to Astute Medical, Inc., San vasopressor required). Diego, Calif. The study urine samples are frozen and shipped to Astute Medical, Inc. Exclusion Criteria 0097 known pregnancy; Example 10 institutionalized individuals; previous renal transplantation; Use of Analyte as a Marker for Sepsis known acutely worsening renal function prior to enrollment (0099 Patients from the intensive care unit (ICU) were (e.g., any category of RIFLE criteria); classified as positive or negative for sepsis according to clini received dialysis (either acute or chronic) within 5 days prior cal diagnosis on each day from enrollment to 6 days after. to enrollment or in imminent need of dialysis at the time of 0100 Two cohorts were defined as (Cohort 1) patients enrollment; who had sepsis, and (Cohort 2) patients who did not have known infection with human immunodeficiency virus (HIV) sepsis on any day from enrollment to 6 days after (7 days or a hepatitis virus; total). Both urine and plasma samples from each patient in meets any of the following: Cohorts 1 and 2 were collected on the day of enrollment. The (i) active bleeding with an anticipated need for >4 units PRBC concentrations of the analyte in these samples were measured in a day; by Standard immunoassay methods using commercially US 2015/0293131 A1 Oct. 15, 2015 available assay reagents. A receiver operating characteristic on any of the 5 days prior to enrollment but had septic shock (ROC) curve was generated using the concentrations, and the on any of the Subsequent 7 days (enrollment to 6 days after), performance of the analyte was assessed by the area under the and (Cohort 2) patients who did not have septic shock on any ROC curve (AUC). The two-tailed p-value of the AUC for the of the 5 days prior to enrollment and on any of the Subsequent analyte was calculated, and if the p-value was less than 0.1. 7 days. The urine and plasma sample collections and analyses the AUC was considered statistically significant. using the measured analyte concentrations were the same as Example 11 in Example 3 above. Use of Analyte as a Marker for Progression to Sepsis Example 14 0101 Two cohorts were defined as (Cohort 1) patients who did not have sepsis during the 5 days prior to enrollment Use of Analyte as a Marker for Death with Sepsis but had sepsis on any of the Subsequent 7 days (enrollment to 6 days after), and (Cohort 2) patients who did not have sepsis 0104. Two cohorts were defined as (Cohort 1) patients on any of the 5 days prior to enrollment and on any of the who had sepsis on any day from enrollment to 6 days after and Subsequent 7 days. The urine and plasma sample collections died within 30 days after enrollment, and (Cohort 2) patients and analyses using the measured analyte concentrations were who did not die and patients who died within 30 days after the same as in Example 3 above. enrollment but did not have sepsis on any day from enroll ment to 6 days after. The urine and plasma sample collections Example 12 and analyses using the measured analyte concentrations were Use of Analyte as a Marker for Septic Shock the same as in Example 3 above. 0102) Two cohorts were defined as (Cohort 1) patients Example 15 who had both sepsis and hypotension on the same day (septic shock), and (Cohort 2) patients who did not have septic shock on any day from enrollment to 6 days after. A patient was Use of Analyte as a Marker for Death with Septic classified as having hypotension if his/her systolic blood Shock pressure was below 70 mm Hg. The urine and plasma sample 0105. Two cohorts were defined as (Cohort 1) patients collections and analyses using the measured analyte concen who had septic shock (see Example 5 for definition) on any trations were the same as in Example 3 above. day from enrollment to 6 days after and died within 30 days Example 13 after enrollment, and (Cohort 2) patients who did not die and patients who died within 30 days after enrollment but did not Use of Analyte as a Marker for Progression to Septic have septic shock on any day from enrollment to 6 days after. Shock The urine and plasma sample collections and analyses using 0103) Two cohorts were defined as (Cohort 1) patients the measured analyte concentrations were the same as in who did not have septic shock (see Example 5 for definition) Example 3 above. TABLE 2 Use of Biomarkers in Sepsis

Preferred Name 1 2 3 4 S 6 7 8 9 10 11 12

Insulin-like growth factor-binding protein 6 S S S S S S Clusterin S S S Tumor necrosis factor receptor Superfamily s s s s s member 10B Trefoil factor 3 Thrombospondin-2 Cathepsin S Hepatitis A virus cellular receptor 1 Carbonic anhydrase 9 Protein NOV homolog Interleukin-29 C-X-C motif chemokine 11 Insulin-like growth factor-binding protein 7 C-X-C motif chemokine 16 C-C motif chemokine 15 Galectin-3 Transmembrane glycoprotein NMB C-C motif chemokine 18 Angiopoietin-related protein 4 Glucagon Cancer Antigen 15-3 Endostatin Angiopoietin-related protein 6 S Epidermal growth factor receptor S Nidogen-1 S Apollipoprotein (a) US 2015/0293131 A1 Oct. 15, 2015 16

TABLE 2-continued Use of Biomarkers in Sepsis Preferred Name 1 2 3 4 S 6 7 8 9 10 11 12 Carcinoembryonic antigen-related cell adhesion S S S S S S molecule 1 Angiopoietin-1 receptor S S S S S Poly ADP-ribose polymerase 1 (cleaved) S S Lymphatic vessel endothelial hyaluronic acid S S S S S receptor 1 Tumor necrosis factor receptor Superfamily S S S member 8 Collagenase 3 S S S C-C motif chemokine 20 S S S S Carcinoembryonic antigen-related cell adhesion S S S molecule 5 Glucagon-like peptide 1 S S S C-C motif chemokine 17 S S S S NF-kappa-B inhibitor alpha S S S Vascular endothelial growth factor D Metalloproteinase inhibitor 3 S S S Osteocalcin S S S S Thymic stromal lymphopoietin S S S S Vascular endothelial growth factor receptor 2 S S S Involucrin S S S S Protransforming growth factor alpha S S S S S P-selectinglycoprotein ligand 1 S S S S Insulin S S Neuronal cell adhesion molecule S S Apollipoprotein C-III S S S S SL cytokine S S Heparin-binding growth factor 1 S S Angiogenin S S Epiregulin S S S Bone morphogenetic protein 7 S S S S Glutathione S-transferase P S Cancer Antigen 19-9 Apollipoprotein A-II S Epithelial cell adhesion molecule S S Cadherin-3 S S S S Tenascin S C-Peptide S S C-C motif chemokine 21 S S Angiopoietin-related protein 3 S S Interleukin-21 S Myeloid differentiation primary response protein S MyD88 Vascular endothelial growth factor receptor 3 S Islet amyloid polypeptide S Insulin-like growth factor 1 receptor S S Macrophage metalloelastase S Fibroblast growth factor 19 S Key for the 12 different uses of the biomarkers in Table 1: 1= use of analyte in urine as a marker for sepsis 2 = use of analyte in urine as a marker for progression to sepsis 3 = use of analyte in urine as a marker for septic shock 4 = use of analyte in urine as a marker for progression to septic shock 5 = use of analyte in urine as a marker for death with sepsis 6 = use of analyte in urine as a marker for death with septic shock 7 = use of analyte in plasma as a marker for sepsis 8 = use of analyte in plasma as a marker for progression to sepsis 9 = use of analyte in plasma as a marker for septic shock 10 = use of analyte in plasma as a marker for progression to septic shock 11 = use of analyte in plasma as a marker for death with sepsis 12 = use of analyte in plasma as a marker for death with septic shock S = two-tailed p-value < 0.10 for AUC

0106 While the invention has been described and exem Modifications therein and other uses will occur to those plified in sufficient detail for those skilled in this art to make skilled in the art. These modifications are encompassed within the spirit of the invention and are defined by the scope and use it, various alternatives, modifications, and improve of the claims. ments should be apparent without departing from the spirit 0107. It will be readily apparent to a person skilled in the and scope of the invention. The examples provided herein are art that varying Substitutions and modifications may be made representative of preferred embodiments, are exemplary, and to the invention disclosed herein without departing from the are not intended as limitations on the scope of the invention. Scope and spirit of the invention. US 2015/0293131 A1 Oct. 15, 2015

0108 All patents and publications mentioned in the speci chemokine 11, C X-C motif chemokine 16, Cyclin fication are indicative of the levels of those of ordinary skill in dependent kinase inhibitor 1, DDRGK domain-contain the art to which the invention pertains. All patents and publi ing protein 1 (DDRGK disclosed as SEQ ID NO: 1), cations are herein incorporated by reference to the same Endostatin, Epidermal growth factor receptor, Epiregu extent as if each individual publication was specifically and lin, Epithelial cell adhesion molecule, Erythropoietin individually indicated to be incorporated by reference. receptor, Fibroblast growth factor 19, Fibroblast growth 0109 The invention illustratively described herein suit factor 23, Galectin-3, Gastric inhibitory polypeptide, ably may be practiced in the absence of any element or ele Glucagon, Glucagon-like peptide 1. Glutathione ments, limitation or limitations which is not specifically dis S-transferase P, Heat shock protein beta-1, Heparin closed herein. Thus, for example, in each instance herein any binding growth factor 1, Hepatitis A virus cellular recep of the terms "comprising”, “consisting essentially of and tor 1, Hepatocyte growth factor receptor, Insulin, Insu “consisting of may be replaced with either of the other two lin-like growth factor 1 receptor. Insulin-like growth terms. The terms and expressions which have been employed factor-binding protein 6. Insulin-like growth factor are used as terms of description and not of limitation, and binding protein 7, Interferon alpha-2. Interleukin-20, there is no intention that in the use of Such terms and expres Interleukin-21, Interleukin-28A, Interleukin-29, Inter sions of excluding any equivalents of the features shown and leukin-33, Involucrin, Islet amyloid polypeptide, Kera described orportions thereof, but it is recognized that various tin, type II cytoskeletal 1 (Keratin-1, -10 mix), Lym modifications are possible within the scope of the invention phatic vessel endothelial hyaluronic acid receptor 1, claimed. Thus, it should be understood that although the Macrophage metalloelastase, Matrix metalloproteinase present invention has been specifically disclosed by preferred 9:Metalloproteinase inhibitor 2 complex, Metallopro embodiments and optional features, modification and varia teinase inhibitor 3, Myeloid differentiation primary tion of the concepts herein disclosed may be resorted to by response protein MyD88, Netrin-1, Netrin-4, Neuronal those skilled in the art, and that Such modifications and varia cell adhesion molecule, NF-kappa-B inhibitor alpha, tions are considered to be within the scope of this invention as Nidogen-1, Osteocalcin, Pappalysin-1, Poly ADP-ri defined by the appended claims. bose polymerase 1 (cleaved), Probetacellulin, Prostate 0110. Other embodiments are set forth within the follow specific antigen, Prostatic acid phosphatase, Protein ing claims. NOV homolog, Protransforming growth factor alpha,

SEQUENCE LISTING

<16O is NUMBER OF SEO ID NOS : 1

<210s, SEQ ID NO 1 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 1 Asp Asp Arg Gly Lys 1. 5

We claim: P-selectinglycoprotein ligand 1, Sex hormone-binding 1. A method of diagnosing SIRS, sepsis, severe sepsis, globulin, SL cytokine, Tenascin, Thrombospondin-2, septic shock, or MODS in a Subject, or assigning a prognostic Thymic stromal lymphopoietin, Transmembrane glyco risk for one or more clinical outcomes for a Subject suffering protein NMB, Trefoil factor 3, Tubulointerstitial nephri from SIRS, sepsis, severe sepsis, septic shock, or MODS, the tis antigen, Tumor necrosis factor receptor Superfamily method comprising: member 10B, Tumor necrosis factor receptor superfam performing one or more assays configured to detect one or ily member 8, Versican core protein, Bone morphoge more biomarkers selected from the group consisting of netic protein 7, Carbonic anhydrase 9, Caspase-9, Col Alpha-2 macroglobulin, Angiogenin, Angiopoietin-1 lagenase 3, Granzyme M, Heparin-binding EGF-like receptor, Angiopoietin-related protein 3, Angiopoietin growth factor, Insulin receptor Substrate 1, Keratin, type related protein 4, Angiopoietin-related protein 6, Apoli II cytoskeletal 6 (6A, -6B, -6C mix), Myeloid differen poprotein A-II, Apollipoprotein C-III, Apollipoprotein tiation primary response protein MyD88, SL cytokine, (a), Bone morphogenetic protein 7. Cadherin-16, Vascular endothelial growth factor D.Vascular endothe Cadherin-3, Cancer Antigen 15-3, Cancer Antigen 19-9., lial growth factor receptor 2, and Vascular endothelial Carbonic anhydrase 9, Carcinoembryonic antigen-re growth factor receptor 3 on a body fluid sample obtained lated cell adhesion molecule 1, Carcinoembryonic anti from the sepsis patient to provide one or more assay gen-related cell adhesion molecule 5. Caspase-3, active, result(s); and Cathepsin S, C-C motif chemokine 1, C-C motif relating the immunoassay result(s) to one or more diag chemokine 15, C-C motif chemokine 17, C-C motif noses or prognoses selected from the group consisting of chemokine 18, C-C motif chemokine 20, C-C motif the presence or absence of SIRS, the presence or absence chemokine 21, C-C motif chemokine 24, Clusterin, of sepsis, the presence or absence of severe sepsis, the Collagenase 3, C-peptide (Insulin), C X-C motif presence or absence of Septic shock, the presence or US 2015/0293131 A1 Oct. 15, 2015 18

absence of MODS, and the prognostic risk of one or 14. A method according to claim 1, wherein the method is more clinical outcomes for the Subject Suffering from or a diagnostic method, and the method differentiates between a believed to suffer from SIRS, sepsis, severe sepsis, sep diagnosis of sepsis and a diagnosis of severe sepsis or septic tic shock, or MODS. shock. 2. A method according to claim 1, wherein the relating step 15. A method according to claim 1, wherein the method is comprises relating the immunoassay result to a prognostic a diagnostic method, and the method differentiates between a risk of mortality. diagnosis of sepsis or severe sepsis and a diagnosis of septic 3. A method according to claim 1, wherein the relating step shock. comprises relating the immunoassay result to a prognostic 16. A method for evaluating biomarker levels in a body risk of one or more future changes in renal status. fluid sample, comprising: 4. A method according to claim3, wherein said one or more obtaining a body fluid sample from a subject selected for future changes in renal status comprise one or more of a future evaluation based on a determination that the Subject is at injury to renal function, future reduced renal function, future risk of a future or current diagnosis of sepsis, severe improvement in renal function, and future acute renal failure sepsis, septic shock or MODS; and (ARF). performing one or more analyte binding assays configured 5. A method according to claim 1, wherein said relating to detect one or more biomarkers selected from the step comprises comparing each of the immunoassay result(s) group consisting of Alpha-2 macroglobulin, Angioge to a corresponding predetermined threshold level selected to nin, Angiopoietin-1 receptor, Angiopoietin-related pro provide a sensitivity or specificity of at least 0.7 for the tein 3, Angiopoietin-related protein 4. Angiopoietin-re diagnosis of sepsis, compared to SIRS not progressed to lated protein 6, Apollipoprotein A-II, Apollipoprotein sepsis. C-III, Apollipoprotein(a), Bone morphogenetic protein 6. A method according to claim 1, wherein said relating 7. Cadherin-16, Cadherin-3, Cancer Antigen 15-3, Can step comprises comparing each of the immunoassay result(s) cer Antigen 19-9, Carbonic anhydrase 9, Carcinoembry to a corresponding predetermined threshold level selected to onic antigen-related cell adhesion molecule 1, Carcino provide a sensitivity or specificity of at least 0.7 for the embryonic antigen-related cell adhesion molecule 5. diagnosis of severe sepsis, compared to SIRS not progressed Caspase-3, active, Cathepsin S, C-C motif chemokine to severe sepsis. 1, C-C motif chemokine 15, C-C motif chemokine 17, C-C motif chemokine 18, C-C motif chemokine 7. A method according to claim 1, wherein said relating 20, C C motif chemokine 21, C C motif chemokine step comprises comparing each of the immunoassay result(s) 24, Clusterin, Collagenase 3, C-peptide (Insulin), to a corresponding predetermined threshold level selected to C X—C motif chemokine 11, C X—C motif provide a sensitivity or specificity of at least 0.7 for the chemokine 16, Cyclin-dependent kinase inhibitor 1, diagnosis of septic shock, compared to SIRS not progressed DDRGK domain-containing protein 1 (DDRGK dis to septic shock. closed as SEQID NO: 1), Endostatin, Epidermal growth 8. A method according to claim 1, wherein said relating factor receptor, Epiregulin, Epithelial cell adhesion mol step comprises comparing each of the immunoassay result(s) ecule, Erythropoietin receptor, Fibroblast growth factor to a corresponding predetermined threshold level selected to 19, Fibroblast growth factor 23, Galectin-3, Gastric provide an odds ratio of at least 2 for the prognostic risk of inhibitory polypeptide, Glucagon, Glucagon-like pep mortality. tide 1. Glutathione S-transferase P. Heat shock protein 9. A method according to claim 1, wherein said relating beta-1, Heparin-binding growth factor 1, Hepatitis A step comprises comparing each of the immunoassay result(s) virus cellular receptor 1, Hepatocyte growth factor to a corresponding predetermined threshold level selected to receptor, Insulin, Insulin-like growth factor 1 receptor, provide an odds ratio of at least 2 for the prognostic risk of a Insulin-like growth factor-binding protein 6. Insulin worsening sepsis classification level selected from the group like growth factor-binding protein 7. Interferon alpha-2, consisting of severe sepsis, septic shock, and MODS. Interleukin-20, Interleukin-21, Interleukin-28A, Inter 10. A method according to claim 1, wherein said relating leukin-29, Interleukin-33, Involucrin, Islet amyloid step comprises comparing each of the immunoassay result(s) polypeptide, Keratin, type II cytoskeletal 1 (Keratin-1, to a corresponding predetermined threshold level selected to -10 mix), Lymphatic vessel endothelial hyaluronic acid provide an odds ratio of at least 2 for the prognostic risk of one receptor 1, Macrophage metalloelastase, Matrix metal or more of a future injury to renal function, future reduced loproteinase-9:Metalloproteinase inhibitor 2 complex, renal function, future improvement in renal function, and Metalloproteinase inhibitor 3, Myeloid differentiation future ARF. primary response protein MyD88, Netrin-1, Netrin-4, Neuronal cell adhesion molecule, NF-kappa-B inhibitor 11. A method according to claim 1, wherein the sample is alpha, Nidogen-1, Osteocalcin, Pappalysin-1, Poly selected from the group consisting of blood, serum, and ADP-ribose polymerase 1 (cleaved), Probetacellulin, plasma. Prostate-specific antigen, Prostatic acid phosphatase, 12. A method according to claim 1, wherein the method is Protein NOV homolog, Protransforming growth factor a prognostic method, and the method differentiates between a alpha, P-selectin glycoprotein ligand 1, Sex hormone risk of future sepsis and a risk of future severe sepsis or septic binding globulin, SL cytokine, Tenascin, Thrombospon shock. din-2, Thymic stromal lymphopoietin, Transmembrane 13. A method according to claim 1, wherein the method is glycoprotein NMB, Trefoil factor 3, Tubulointerstitial a prognostic method, and the method differentiates between a nephritis antigen, Tumor necrosis factor receptor Super risk of future sepsis or severe sepsis and a risk of future septic family member 10B, Tumor necrosis factor receptor shock. superfamily member 8, Versican core protein, Bone US 2015/0293131 A1 Oct. 15, 2015 19

morphogenetic protein 7, Carbonic anhydrase 9, C X—C motif chemokine 11, C X—C motif Caspase-9, Collagenase 3, Granzyme M. Heparin-bind chemokine 16, Cyclin-dependent kinase inhibitor 1, ing EGF-like growth factor. Insulin receptor substrate 1, DDRGK domain-containing protein 1 (DDRGK dis Keratin, type II cytoskeletal 6 (6A, -6B, -6C mix), closed as SEQID NO: 1), Endostatin, Epidermal growth Myeloid differentiation primary response protein factor receptor, Epiregulin, Epithelial cell adhesion mol MyD88, SL cytokine, Vascular endothelial growth fac ecule, Erythropoietin receptor, Fibroblast growth factor tor D.Vascular endothelial growth factor receptor 2, and 19, Fibroblast growth factor 23, Galectin-3, Gastric Vascular endothelial growth factor receptor 3 by intro inhibitory polypeptide, Glucagon, Glucagon-like pep ducing the body fluid sample obtained from the subject tide 1. Glutathione S-transferase P. Heat shock protein into an assay instrument which (i) contacts a plurality of beta-1, Heparin-binding growth factor 1, Hepatitis A reagents which specifically bind for detection the plu virus cellular receptor 1, Hepatocyte growth factor rality of biomarkers with the urine sample, and (ii) gen receptor, Insulin, Insulin-like growth factor 1 receptor, erates one or more assay results indicative of binding of Insulin-like growth factor-binding protein 6. Insulin each biomarker which is assayed to a respective specific like growth factor-binding protein 7. Interferon alpha-2, binding reagent in the plurality of reagents; and Interleukin-20, Interleukin-21, Interleukin-28A, Inter displaying the one or more assay results from the assay leukin-29, Interleukin-33, Involucrin, Islet amyloid instrument as a quantitative result in a human-readable polypeptide, Keratin, type II cytoskeletal 1 (Keratin-1, form. -10 mix), Lymphatic vessel endothelial hyaluronic acid 17. A method according to claim 16, wherein the subject is receptor 1, Macrophage metalloelastase, Matrix metal selected for evaluation based on a determination that the loproteinase-9:Metalloproteinase inhibitor 2 complex, Subject has sepsis and is at risk of a future diagnosis of severe Metalloproteinase inhibitor 3, Myeloid differentiation sepsis, septic shock or MODS. primary response protein MyD88, Netrin-1, Netrin-4, 18. A method according to claim 16, wherein the subject is Neuronal cell adhesion molecule, NF-kappa-B inhibitor selected for evaluation based on a determination that the alpha, Nidogen-1, Osteocalcin, Pappalysin-1, Poly Subject has sepsis or severe sepsis and is at risk of a future ADP-ribose polymerase 1 (cleaved), Probetacellulin, diagnosis of septic shock or MODS. Prostate-specific antigen, Prostatic acid phosphatase, 19. A method according to claim 16, wherein a plurality of Protein NOV homolog, Protransforming growth factor assay results are combined using a function that converts said alpha, P-selectin glycoprotein ligand 1, Sex hormone assay results into a single composite result. binding globulin, SL cytokine, Tenascin, Thrombospon 20. A method according to claim 16, wherein the subject is din-2, Thymic stromal lymphopoietin, Transmembrane selected for evaluation based on a determination that the glycoprotein NMB, Trefoil factor 3, Tubulointerstitial Subject is at risk of a future diagnosis of severe sepsis, septic nephritis antigen, Tumor necrosis factor receptor Super shock or MODS within a period selected from the group family member 10B, Tumor necrosis factor receptor consisting of 21 days, 14 days, 7 days, 5 days, 96 hours, 72 superfamily member 8, Versican core protein, Bone hours, 48 hours, 36 hours, 24 hours, and 12 hours. morphogenetic protein 7, Carbonic anhydrase 9, 21. A method according to claim 16, wherein the subject is Caspase-9, Collagenase 3, Granzyme M. Heparin-bind selected for evaluation based on a determination that the ing EGF-like growth factor. Insulin receptor substrate 1, Subject is at risk of a future diagnosis of one or more of an Keratin, type II cytoskeletal 6 (6A, -6B, -6C mix), injury to renal function, reduced renal function, improvement Myeloid differentiation primary response protein in renal function, and future ARF. MyD88, SL cytokine, Vascular endothelial growth fac 22. A method according to claim 16, wherein the plurality tor D.Vascular endothelial growth factor receptor 2, and of assays are immunoassays performed by (i) introducing the Vascular endothelial growth factor receptor 3: urine sample into an assay device comprising a plurality of an assay instrument configured to receive a urine sample antibodies, at least one of which binds to each biomarker and contact the plurality of reagents with the urine which is assayed, and (ii) generatinganassay result indicative sample and to generate and quantitatively display in of binding of each biomarker to its respective antibody. human readable form one or more assay results indica 23. A system for evaluating biomarker levels, comprising: tive of binding of each biomarker which is assayed to a a plurality of reagents which specifically bind for detection respective specific binding reagent in the plurality of the plurality of biomarkers selected from the group con reagents. sisting of Alpha-2 macroglobulin, Angiogenin, Angiopoietin-1 receptor, Angiopoietin-related protein 24. A system according to claim 23 wherein the reagents 3, Angiopoietin-related protein 4, Angiopoietin-related comprise a plurality of antibodies, at least one of which binds protein 6, Apollipoprotein A-II, Apollipoprotein C-Ill. to each of the biomarkers which are assayed. Apollipoprotein(a), Bone morphogenetic protein 7, Cad 25. A system according to claim 23 wherein assay instru herin-16, Cadherin-3, Cancer Antigen 15-3, Cancer ment comprises an assay device and an assay device reader, Antigen 19-9, Carbonic anhydrase 9, Carcinoembryonic wherein the plurality of antibodies are immobilized at a plu antigen-related cell adhesion molecule 1, Carcinoem rality of predetermined locations within the assay device, bryonic antigen-related cell adhesion molecule 5. wherein the assay device is configured to receive the urine Caspase-3, active, Cathepsin S, C-C motif chemokine sample such that the urine sample contacts the plurality of 1, C-C motif chemokine 15, C-C motif chemokine predetermined locations, and wherein the assay device reader 17, C-C motif chemokine 18, C-C motif chemokine interrogates the plurality of predetermined locations to gen 20, C C motif chemokine 21, C-C motif chemokine erate the assay results. 24, Clusterin, Collagenase 3, C-peptide (Insulin), k k k k k