Methods and compositions for diagnosis and prognosis of appendicitis and differentiation of causes of abdominal pain

Abstract

The present invention relates to methods and compositions for monitoring, diagnosis, prognosis, and determination of treatment regimens in appendicitis patients and in patients at risk for appendicitis. In particular, the invention relates to using as-says that detect one or more biomarkers as diagnostic and prognostic biomarker assays in such patients.

We claim: 
     
         1 . A method of diagnosing appendicitis in a subject, or assigning a likelihood of a future outcome to a subject diagnosed with appendicitis, comprising:
 performing one or more assays configured to detect one or more biomarkers selected from the group consisting of 72 kDa type IV collagenase, Adiponectin, Advanced glycosylation end product-specific receptor, Alpha-2 macroglobulin, Alpha-2-HS-glycoprotein, Alpha-fetoprotein, Angiopoietin-1, Antileukoproteinase, Apolipoprotein(a), Cancer Antigen 15-3, Cancer Antigen 19-9, Carbonic anhydrase 9, Carcinoembryonic antigen-related cell adhesion molecule 5, C—C motif chemokine 1, C—C motif chemokine 13, C—C motif chemokine 15, C—C motif chemokine 17, C—C motif chemokine 19, C—C motif chemokine 20, C—C motif chemokine 21, C—C motif chemokine 22, C—C motif chemokine 23, C—C motif chemokine 24, C—C motif chemokine 26, C—C motif chemokine 27, C—C motif chemokine 3, C—C motif chemokine 4, C—C motif chemokine 7, C—C motif chemokine 8, Ceruloplasmin, Choriogonadotropin subunit beta, Collagenase 3, C-Peptide, Creatine Kinase-MB, C—X—C motif chemokine 10, C—X—C motif chemokine 11, C—X—C motif chemokine 13, C—X—C motif chemokine 16, C—X—C motif chemokine 5, C—X—C motif chemokine 6, C—X—C motif chemokine 9, Cystatin-C, Endothelial protein C receptor, Eotaxin, Epidermal growth factor receptor, Ferritin, Fibrinogen, Gastric inhibitory polypeptide, Glucagon, Glucagon-like peptide 1, Granulocyte-macrophage colony-stimulating factor, Growth-regulated alpha, beta, and gamma proteins, Heparin-binding growth factor 2, Hepatocyte growth factor, Immuno globulin A, Immunoglobulin M, Immunoglogulin G1, Immunoglogulin G2, Immunoglogulin G3, Immunoglogulin G4, Insulin, Insulin-like growth factor-binding protein 1, Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, Insulin-like growth factor-binding protein 5, Insulin-like growth factor-binding protein 6, Insulin-like growth factor-binding protein 7, Interferon alpha-2, Interferon gamma, Interleukin-1 alpha, Interleukin-1 beta, Interleukin-1 receptor antagonist protein, Interleukin-1 receptor type I, Interleukin-1 receptor type II, Interleukin-11, Interleukin-12, Interleukin-12 subunit beta, Interleukin-13, Interleukin-15, Interleukin-2, Interleukin-20, Interleukin-21, Interleukin-23, Interleukin-28A, Interleukin-29, Interleukin-3, Interleukin-33, Interleukin-4, Interleukin-5, Interleukin-6 receptor subunit alpha, Interleukin-6 receptor subunit beta, Interleukin-7, Interleukin-9, Interstitial collagenase, Islet amyloid polypeptide, Keratin, type I cytoskeletal 19 (aa311-367), Kit ligand, Leptin, Leukemia inhibitory factor, Lymphotactin, Lymphotoxin-alpha, Macrophage colony-stimulating factor 1, Macrophage metalloelastase, Macrophage migration inhibitory factor, Matrilysin, Metalloproteinase inhibitor 1, Metalloproteinase inhibitor 2, Metalloproteinase inhibitor 3, Metalloproteinase inhibitor 4, Myoglobin, Pancreatic prohormone, Peptide YY, Pro-epidermal growth factor, Pro-interleukin-16, Prolactin, Prostate-specific antigen, Protein S100-A12, Protransforming growth factor alpha, Secretory immunoglobulin A, Serum amyloid P-component, SL cytokine, Stromal cell-derived factor 1, Stromelysin-1, Thrombopoietin, Thymic stromal lymphopoietin, Tumor necrosis factor, Tumor necrosis factor ligand superfamily member 10, Tumor necrosis factor ligand superfamily member 6, Tumor necrosis factor receptor superfamily member 1A, Tumor necrosis factor receptor superfamily member 1B, Vascular endothelial growth factor receptor 1, Vascular endothelial growth factor receptor 2, Vascular endothelial growth factor receptor 3, von Willebrand Factor, and WAP four-disulfide core domain protein 2 on a body fluid sample obtained from the subject to provide one or more assay result(s); and   correlating the assay result(s) to the occurrence or nonoccurrence of appendicitis in the subject or likelihood of the future outcome to the subject.   
     
     
         2 . A method according to  claim 1 , wherein the performing step comprises introducing the body fluid sample obtained from the subject into an assay instrument which (i) contacts the body fluid sample with one or more binding reagents corresponding to the biomarker(s) being assayed, wherein each biomarker which is assayed binds to its respective specific binding reagent in an amount related to its concentration in the body fluid sample, (ii) generates one or more assay results indicative of binding of each biomarker which is assayed to its respective specific binding reagent; and (iii) displays the one or more assay results as a quantitative result in a human-readable form. 
     
     
         3 . A method according to  claim 1  or  2 , wherein the future outcome is mortality. 
     
     
         4 . A method according to  claim 1  or  2 , wherein the subject is being evaluated for abdominal pain. 
     
     
         5 . A method according to  claim 1  or  2 , wherein the correlating step comprises determining the concentration of each biomarker which is assayed, and individually comparing each biomarker concentration to a corresponding threshold level for that biomarker. 
     
     
         6 . A method according to  claim 5 , wherein the assay instrument performs the correlating step, which comprises determining the concentration of each biomarker which is assayed, individually comparing each biomarker concentration to a corresponding threshold level for that biomarker, and displaying an indication of whether each biomarker does or does not exceed its corresponding threshold in a human-readable form. 
     
     
         7 . A method according to  claim 2 , wherein a plurality of the biomarkers are measured, wherein the assay instrument performs the correlating step, which comprises determining the concentration of each of the plurality of biomarkers, calculating a single value based on the concentration of each of the plurality of biomarkers, comparing the single value to a corresponding threshold level and displaying an indication of whether the single value does or does not exceed its corresponding threshold in a human-readable form. 
     
     
         8 . A method according to one of  claims 1 - 7 , wherein method provides a sensitivity or specificity of at least 0.7 for the identification of appendicitis when compared to normal subjects. 
     
     
         9 . A method according to one of  claims 1 - 7 , wherein method provides a sensitivity or specificity of at least 0.7 for the identification of appendicitis when compared to subjects exhibiting symptoms that mimic appendicitis symptoms. 
     
     
         10 . A method according to one of  claims 1 - 9 , wherein the sample is selected from the group consisting of blood, serum, and plasma. 
     
     
         11 . A method for evaluating biomarker levels in a body fluid sample, comprising:
 obtaining a body fluid sample from a subject selected for evaluation based on a determination that the subject is experiencing symptoms indicative of possible acute appendicitis; and   performing one or more analyte binding assays configured to detect one or more biomarkers selected from the group consisting of 72 kDa type IV collagenase, Adiponectin, Advanced glycosylation end product-specific receptor, Alpha-2 macroglobulin, Alpha-2-HS-glycoprotein, Alpha-fetoprotein, Angiopoietin-1, Antileukoproteinase, Apolipoprotein(a), Cancer Antigen 15-3, Cancer Antigen 19-9, Carbonic anhydrase 9, Carcinoembryonic antigen-related cell adhesion molecule 5, C—C motif chemokine 1, C—C motif chemokine 13, C—C motif chemokine 15, C—C motif chemokine 17, C—C motif chemokine 19, C—C motif chemokine 20, C—C motif chemokine 21, C—C motif chemokine 22, C—C motif chemokine 23, C—C motif chemokine 24, C—C motif chemokine 26, C—C motif chemokine 27, C—C motif chemokine 3, C—C motif chemokine 4, C—C motif chemokine 7, C—C motif chemokine 8, Ceruloplasmin, Choriogonadotropin subunit beta, Collagenase 3, C-Peptide, Creatine Kinase-MB, C—X—C motif chemokine 10, C—X—C motif chemokine 11, C—X—C motif chemokine 13, C—X—C motif chemokine 16, C—X—C motif chemokine 5, C—X—C motif chemokine 6, C—X—C motif chemokine 9, Cystatin-C, Endothelial protein C receptor, Eotaxin, Epidermal growth factor receptor, Ferritin, Fibrinogen, Gastric inhibitory polypeptide, Glucagon, Glucagon-like peptide 1, Granulocyte-macrophage colony-stimulating factor, Growth-regulated alpha, beta, and gamma proteins, Heparin-binding growth factor 2, Hepatocyte growth factor, Immunoglobulin A, Immunoglobulin M, Immunoglogulin G1, Immunoglogulin G2, Immunoglogulin G3, Immunoglogulin G4, Insulin, Insulin-like growth factor-binding protein 1, Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, Insulin-like growth factor-binding protein 5, Insulin-like growth factor-binding protein 6, Insulin-like growth factor-binding protein 7, Interferon alpha-2, Interferon gamma, Interleukin-1 alpha, Interleukin-1 beta, Interleukin-1 receptor antagonist protein, Interleukin-1 receptor type I, Interleukin-1 receptor type II, Interleukin-11, Interleukin-12, Interleukin-12 subunit beta, Interleukin-13, Interleukin-15, Interleukin-2, Interleukin-20, Interleukin-21, Interleukin-23, Interleukin-28A, Interleukin-29, Interleukin-3, Interleukin-33, Interleukin-4, Interleukin-5, Interleukin-6 receptor subunit alpha, Interleukin-6 receptor subunit beta, Interleukin-7, Interleukin-9, Interstitial collagenase, Islet amyloid polypeptide, Keratin, type I cytoskeletal 19 (aa311-367), Kit ligand, Leptin, Leukemia inhibitory factor, Lymphotactin, Lymphotoxin-alpha, Macrophage colony-stimulating factor 1, Macrophage metalloelastase, Macrophage migration inhibitory factor, Matrilysin, Metalloproteinase inhibitor 1, Metalloproteinase inhibitor 2, Metalloproteinase inhibitor 3, Metalloproteinase inhibitor 4, Myoglobin, Pancreatic prohormone, Peptide YY, Pro-epidermal growth factor, Pro-interleukin-16, Prolactin, Prostate-specific antigen, Protein S100-A12, Protransforming growth factor alpha, Secretory immunoglobulin A, Serum amyloid P-component, SL cytokine, Stromal cell-derived factor 1, Stromelysin-1, Thrombopoietin, Thymic stromal lymphopoietin, Tumor necrosis factor, Tumor necrosis factor ligand superfamily member 10, Tumor necrosis factor ligand superfamily member 6, Tumor necrosis factor receptor superfamily member 1A, Tumor necrosis factor receptor superfamily member 1B, Vascular endothelial growth factor receptor 1, Vascular endothelial growth factor receptor 2, Vascular endothelial growth factor receptor 3, von Willebrand Factor, and WAP four-disulfide core domain protein 2 by introducing the body fluid sample obtained from the subject into an assay instrument which (i) contacts the body fluid sample with one or more binding reagents corresponding to the biomarker(s) being assayed, wherein each biomarker which is assayed binds to its respective specific binding reagent in an amount related to its concentration in the body fluid sample, (ii) generates one or more assay results indicative of binding of each biomarker which is assayed to its respective specific binding reagent; and (iii) displays the one or more assay results as a quantitative result in a human-readable form.   
     
     
         12 . A method according to  claim 11 , wherein the assay result(s) are displayed as a concentration of each biomarker which is assayed. 
     
     
         13 . A method according to  claim 12 , wherein the assay instrument further individually compares each biomarker concentration to a corresponding threshold level for that biomarker, and displays an indication of whether each biomarker does or does not exceed its corresponding threshold in a human-readable form. 
     
     
         14 . A method according to  claim 11 , wherein a plurality of the biomarkers are measured, and wherein the assay results(s) comprise a single value calculated using a function that converts the concentration of each of the plurality of biomarkers into a single value. 
     
     
         15 . A method according to  claim 14 , wherein the assay instrument further compares the single value to a corresponding threshold level and displays an indication of whether the single value does or does not exceed its corresponding threshold in a human-readable form. 
     
     
         16 . A method for evaluating biomarker levels in a body fluid sample, comprising:
 obtaining a body fluid sample from a subject selected for evaluation based on a determination that the subject has been diagnosed with acute appendicitis; and   performing one or more analyte binding assays configured to detect one or more biomarkers selected from the group consisting of 72 kDa type IV collagenase, Adiponectin, Advanced glycosylation end product-specific receptor, Alpha-2 macroglobulin, Alpha-2-HS-glycoprotein, Alpha-fetoprotein, Angiopoietin-1, Antileukoproteinase, Apolipoprotein(a), Cancer Antigen 15-3, Cancer Antigen 19-9, Carbonic anhydrase 9, Carcinoembryonic antigen-related cell adhesion molecule 5, C—C motif chemokine 1, C—C motif chemokine 13, C—C motif chemokine 15, C—C motif chemokine 17, C—C motif chemokine 19, C—C motif chemokine 20, C—C motif chemokine 21, C—C motif chemokine 22, C—C motif chemokine 23, C—C motif chemokine 24, C—C motif chemokine 26, C—C motif chemokine 27, C—C motif chemokine 3, C—C motif chemokine 4, C—C motif chemokine 7, C—C motif chemokine 8, Ceruloplasmin, Choriogonadotropin subunit beta, Collagenase 3, C-Peptide, Creatine Kinase-MB, C—X—C motif chemokine 10, C—X—C motif chemokine 11, C—X—C motif chemokine 13, C—X—C motif chemokine 16, C—X—C motif chemokine 5, C—X—C motif chemokine 6, C—X—C motif chemokine 9, Cystatin-C, Endothelial protein C receptor, Eotaxin, Epidermal growth factor receptor, Ferritin, Fibrinogen, Gastric inhibitory polypeptide, Glucagon, Glucagon-like peptide 1, Granulocyte-macrophage colony-stimulating factor, Growth-regulated alpha, beta, and gamma proteins, Heparin-binding growth factor 2, Hepatocyte growth factor, Immunoglobulin A, Immunoglobulin M, Immunoglogulin G1, Immunoglogulin G2, Immunoglogulin G3, Immunoglogulin G4, Insulin, Insulin-like growth factor-binding protein 1, Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, Insulin-like growth factor-binding protein 5, Insulin-like growth factor-binding protein 6, Insulin-like growth factor-binding protein 7, Interferon alpha-2, Interferon gamma, Interleukin-1 alpha, Interleukin-1 beta, Interleukin-1 receptor antagonist protein, Interleukin-1 receptor type I, Interleukin-1 receptor type II, Interleukin-11, Interleukin-12, Interleukin-12 subunit beta, Interleukin-13, Interleukin-15, Interleukin-2, Interleukin-20, Interleukin-21, Interleukin-23, Interleukin-28A, Interleukin-29, Interleukin-3, Interleukin-33, Interleukin-4, Interleukin-5, Interleukin-6 receptor subunit alpha, Interleukin-6 receptor subunit beta, Interleukin-7, Interleukin-9, Interstitial collagenase, Islet amyloid polypeptide, Keratin, type I cytoskeletal 19 (aa311-367), Kit ligand, Leptin, Leukemia inhibitory factor, Lymphotactin, Lymphotoxin-alpha, Macrophage colony-stimulating factor 1, Macrophage metalloelastase, Macrophage migration inhibitory factor, Matrilysin, Metalloproteinase inhibitor 1, Metalloproteinase inhibitor 2, Metalloproteinase inhibitor 3, Metalloproteinase inhibitor 4, Myoglobin, Pancreatic prohormone, Peptide YY, Pro-epidermal growth factor, Pro-interleukin-16, Prolactin, Prostate-specific antigen, Protein S100-A12, Protransforming growth factor alpha, Secretory immunoglobulin A, Serum amyloid P-component, SL cytokine, Stromal cell-derived factor 1, Stromelysin-1, Thrombopoietin, Thymic stromal lymphopoietin, Tumor necrosis factor, Tumor necrosis factor ligand superfamily member 10, Tumor necrosis factor ligand superfamily member 6, Tumor necrosis factor receptor superfamily member 1A, Tumor necrosis factor receptor superfamily member 1B, Vascular endothelial growth factor receptor 1, Vascular endothelial growth factor receptor 2, Vascular endothelial growth factor receptor 3, von Willebrand Factor, and WAP four-disulfide core domain protein 2 by introducing the body fluid sample obtained from the subject into an assay instrument which (i) contacts the body fluid sample with one or more binding reagents corresponding to the biomarker(s) being assayed, wherein each biomarker which is assayed binds to its respective specific binding reagent in an amount related to its concentration in the body fluid sample, (ii) generates one or more assay results indicative of binding of each biomarker which is assayed to its respective specific binding reagent; and (iii) displays the one or more assay results as a quantitative result in a human-readable form.   
     
     
         17 . A method according to  claim 16 , wherein the assay result(s) are displayed as a concentration of each biomarker which is assayed. 
     
     
         18 . A method according to  claim 17 , wherein the assay instrument further individually compares each biomarker concentration to a corresponding threshold level for that biomarker, and displays an indication of whether each biomarker does or does not exceed its corresponding threshold in a human-readable form. 
     
     
         19 . A method according to  claim 16 , wherein a plurality of the biomarkers are measured, and wherein the assay results(s) comprise a single value calculated using a function that converts the concentration of each of the plurality of biomarkers into a single value. 
     
     
         20 . A method according to  claim 19 , wherein the assay instrument further compares the single value to a corresponding threshold level and displays an indication of whether the single value does or does not exceed its corresponding threshold in a human-readable form. 
     
     
         21 . A method according to one of  claims 16 - 19 , wherein the subject is selected for evaluation of a mortality risk within a period selected from the group consisting of 21 days, 14 days, 7 days, 5 days, 96 hours, 72 hours, 48 hours, 36 hours, 24 hours, and 12 hours. 
     
     
         22 . A method according to one of  claims 11 - 21 , wherein the plurality of assays are immunoassays performed by (i) introducing the body fluid sample into an assay device comprising a plurality of antibodies, at least one of which binds to each biomarker which is assayed, and (ii) generating an assay result indicative of binding of each biomarker to its respective antibody. 
     
     
         23 . A system for evaluating biomarker levels, comprising:
 a plurality of reagents which specifically bind for detection a plurality of biomarkers selected from the group consisting of 72 kDa type IV collagenase, Adiponectin, Advanced glycosylation end product-specific receptor, Alpha-2 macroglobulin, Alpha-2-HS-glycoprotein, Alpha-fetoprotein, Angiopoietin-1, Antileukoproteinase, Apolipoprotein(a), Cancer Antigen 15-3, Cancer Antigen 19-9, Carbonic anhydrase 9, Carcinoembryonic antigen-related cell adhesion molecule 5, C—C motif chemokine 1, C—C motif chemokine 13, C—C motif chemokine 15, C—C motif chemokine 17, C—C motif chemokine 19, C—C motif chemokine 20, C—C motif chemokine 21, C—C motif chemokine 22, C—C motif chemokine 23, C—C motif chemokine 24, C—C motif chemokine 26, C—C motif chemokine 27, C—C motif chemokine 3, C—C motif chemokine 4, C—C motif chemokine 7, C—C motif chemokine 8, Ceruloplasmin, Choriogonadotropin subunit beta, Collagenase 3, C-Peptide, Creatine Kinase-MB, C—X—C motif chemokine 10, C—X—C motif chemokine 11, C—X—C motif chemokine 13, C—X—C motif chemokine 16, C—X—C motif chemokine 5, C—X—C motif chemokine 6, C—X—C motif chemokine 9, Cystatin-C, Endothelial protein C receptor, Eotaxin, Epidermal growth factor receptor, Ferritin, Fibrinogen, Gastric inhibitory polypeptide, Glucagon, Glucagon-like peptide 1, Granulocyte-macrophage colony-stimulating factor, Growth-regulated alpha, beta, and gamma proteins, Heparin-binding growth factor 2, Hepatocyte growth factor, Immunoglobulin A, Immunoglobulin M, Immunoglogulin G1, Immunoglogulin G2, Immunoglogulin G3, Immunoglogulin G4, Insulin, Insulin-like growth factor-binding protein 1, Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, Insulin-like growth factor-binding protein 5, Insulin-like growth factor-binding protein 6, Insulin-like growth factor-binding protein 7, Interferon alpha-2, Interferon gamma, Interleukin-1 alpha, Interleukin-1 beta, Interleukin-1 receptor antagonist protein, Interleukin-1 receptor type I, Interleukin-1 receptor type II, Interleukin-11, Interleukin-12, Interleukin-12 subunit beta, Interleukin-13, Interleukin-15, Interleukin-2, Interleukin-20, Interleukin-21, Interleukin-23, Interleukin-28A, Interleukin-29, Interleukin-3, Interleukin-33, Interleukin-4, Interleukin-5, Interleukin-6 receptor subunit alpha, Interleukin-6 receptor subunit beta, Interleukin-7, Interleukin-9, Interstitial collagenase, Islet amyloid polypeptide, Keratin, type I cytoskeletal 19 (aa311-367), Kit ligand, Leptin, Leukemia inhibitory factor, Lymphotactin, Lymphotoxin-alpha, Macrophage colony-stimulating factor 1, Macrophage metalloelastase, Macrophage migration inhibitory factor, Matrilysin, Metalloproteinase inhibitor 1, Metalloproteinase inhibitor 2, Metalloproteinase inhibitor 3, Metalloproteinase inhibitor 4, Myoglobin, Pancreatic prohormone, Peptide YY, Pro-epidermal growth factor, Pro-interleukin-16, Prolactin, Prostate-specific antigen, Protein S100-A12, Protransforming growth factor alpha, Secretory immunoglobulin A, Serum amyloid P-component, SL cytokine, Stromal cell-derived factor 1, Stromelysin-1, Thrombopoietin, Thymic stromal lymphopoietin, Tumor necrosis factor, Tumor necrosis factor ligand superfamily member 10, Tumor necrosis factor ligand superfamily member 6, Tumor necrosis factor receptor superfamily member 1A, Tumor necrosis factor receptor superfamily member 1B, Vascular endothelial growth factor receptor 1, Vascular endothelial growth factor receptor 2, Vascular endothelial growth factor receptor 3, von Willebrand Factor, and WAP four-disulfide core domain protein 2;   an assay instrument configured to (i) receive a body fluid sample, (ii) contact the plurality of reagents with the body fluid sample and (iii) generate and quantitatively display in human readable form one or more assay results indicative of binding of each biomarker which is assayed to a respective specific binding reagent in the plurality of reagents.   
     
     
         24 . A system according to  claim 23  wherein the reagents comprise a plurality of antibodies, at least one of which binds to each of the biomarkers which are assayed. 
     
     
         25 . A system according to  claim 24  wherein assay instrument comprises an assay device and an assay device reader, wherein the plurality of antibodies are immobilized at a plurality of predetermined locations within the assay device, wherein the assay device is configured to receive the body fluid sample such that the body fluid sample contacts the plurality of predetermined locations, and wherein the assay device reader interrogates the plurality of predetermined locations to generate the assay results.