WO2012071516A2 - Devices and methods for the diagnosis and treatment of wounds using biomarkers - Google Patents
Devices and methods for the diagnosis and treatment of wounds using biomarkers Download PDFInfo
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- WO2012071516A2 WO2012071516A2 PCT/US2011/062049 US2011062049W WO2012071516A2 WO 2012071516 A2 WO2012071516 A2 WO 2012071516A2 US 2011062049 W US2011062049 W US 2011062049W WO 2012071516 A2 WO2012071516 A2 WO 2012071516A2
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/475—Assays involving growth factors
- G01N2333/50—Fibroblast growth factors [FGF]
- G01N2333/503—Fibroblast growth factors [FGF] basic FGF [bFGF]
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/908—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/966—Elastase
Definitions
- the present invention relates generally to the field of biology. More particularly, it relates to devices and methods for the diagnosis and treatment of wounds using biomarkers.
- the present invention provides for devices and methods for determining whether a wound is within the normal trajectory of healing or to identify the stage where it has arrested, without causing further damage to the patient.
- the present invention provides a wound diagnosis device comprising a surface and at least one agent that is specific to a desired biomarker.
- the device comprises a solid surface having at least a first agent that is specific to a first biomarker and a least a second agent that is specific to a second biomarker.
- the device further comprises at least a third agent that is specific to a third biomarker.
- the device comprises 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70 80, 90, 100, or more agents that are specific to 1 , 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70 80, 90, 100, or more biomarkers.
- the surface may be any surface to which the desired agents may be attached, including but not limited to a wound dressing or a microarray.
- the surface is an adsorbant surface.
- the adsorbant surface comprises filter paper or colorimetric paper.
- the device is a wound dressing.
- the wound dressing may be any appropriate type of wound dressing.
- the wound dressing comprises a polyurethane film dressing.
- the surface is a microarray.
- the agent may be any appropriate agent that is specific to a desired biomarker.
- the agent may be aptamers, ligands, antibodies, or peptide sequences.
- the agents may be a combination of aptamers, ligands, antibodies, and/or peptide sequences in any combination.
- the agent may be attached to the surface in any appropriate manner.
- the device is coated or bound with agents that bind to the biomarkers.
- the agents may be bound chemically (ionic or covalently) or physically to the wound dressing.
- the agents are bound by chemical grafting, polymer- binder, bi- functional chemical binding (e.g., silane or pluronic), hydrophilic or electrochemical binding, or plasma coating.
- the device may be coated with the agents.
- the agents are linked to the solid surface by covalent bonds.
- the biomarker may be any secreted biomarker.
- the biomarker may be myeloperoxidase (MPO), neutrophil elastase (nElastase), Human Neutrophil Lipolin (HNL), Lactoferrin, Lysozyme, Neutrophil Gelatinase-Associated Lipocalin (NGAL), Human Neutrophil Elastase Anti-Neutrophil Cytoplasmic Antibodies (HNE ANCA's), MMP9, Proteinase 3, Serpin Peptidase Inhibitor Clade B and D, Reactive Oxygen Species (ROS), or Reactive Nitrogen Species (RNS), or any combination thereof.
- MPO myeloperoxidase
- nElastase neutrophil elastase
- HNL Human Neutrophil Lipolin
- NGAL Neutrophil Gelatinase-Associated Lipocalin
- HNE ANCA's Human Neutrophil Elastase Anti-Neutrophil Cyto
- the biomarker may be basic fibroblast growth factor (FGF-2), Fibroblast Growth Factor- 10 (FGF- 10), Fibroblast-specific protein 1 (FSP1), prolyl-4-hydroxylase (5B5), Insulin Growth Factor- 1 (IGF-1), Tetranectin, Collagen alpha 1 , 2, and 3 chains, SERPINAl , or Compliment Components or any combination thereof.
- FGF-2 basic fibroblast growth factor
- FGF- 10 Fibroblast Growth Factor- 10
- FSP1 Fibroblast-specific protein 1
- IGF-1 Insulin Growth Factor- 1
- Tetranectin Collagen alpha 1 , 2, and 3 chains
- SERPINAl Compliment Components or any combination thereof.
- the biomarker may be calgranulin A/B, Cystatin A, S 100 Calcium Binding Proteins, CD163, CD204, CD206, AM-3K, CSF-1R (colony- stimulating factor- 1 receptor), a specific marker of macrophages, EMR1 (epidermal growth factor module-containing mucin-like receptor 1), F4/80, pro-collagen, collagen, or fibronectin or any combination thereof. Any of these biomarkers may be used alone or in combination.
- the first biomarker is MPO and the second biomarker is
- the first biomarker is nElastase and the second biomarker is FGF-10.
- the first biomarker is calgranulin A/B, TNF-a, MPO, or nElastase and the second biomarker is CD204 or CD206.
- the first biomarker is pro-collagen or collagen and the second biomarker is fibronectin.
- the present invention provides a method of determining the phase of wound healing comprising: a) detecting the amount of a first biomarker and the amount of a second biomarker in a sample from a wound; and b) comparing the amount of the first biomarker and the amount of the second biomarker, wherein the relation between the amount of the first biomarker to the amount of the second biomarker indicates the phase of wound healing.
- the sample that is analyzed by the devices and methods disclosed herein may be from any desirable source.
- the sample may be from a wound exudate.
- the sample is wound fluid.
- the sample may be collected from other sources.
- the biomarkers may be extracted from tissue which is either adhered to dressing material (upon removal of the dressing from the wound) or from non-necrotic tissue removed during debridement of the wound.
- the first biomarker has been secreted from neutrophil cells and the second biomarker has been secreted from fibroblast cells. In some embodiments, if the amount of the first biomarker is greater than the amount of the second biomarker, the inflammatory phase of wound healing is indicated. Conversely, if the amount of the first biomarker is less than the amount of the second biomarker, the proliferative phase of wound healing is indicated.
- the first biomarker is MPO and the second biomarker is
- FGF-2 FGF-2.
- the amount of MPO is at least twice greater than the amount of FGF-2, then the inflammatory phase of wound healing is indicated. Conversely, if the amount of MPO is half as much or less than he amount of FGF-2, then the proliferative stage of wound healing is indicated.
- the first biomarker is nElastase and the second biomarker is Fibroblast Growth Factor-10 (FGF-10).
- FGF-10 Fibroblast Growth Factor-10
- the first biomarker has been secreted from Ml macrophage cells and the second biomarker has been secreted from M2 macrophage cells.
- a larger amount of the first biomarker secreted from Ml macrophage cells and a smaller amount of the second biomarker secreted from M2 macrophage cells indicates the inflammatory phase of wound healing.
- a smaller amount of the first biomarker secreted from Ml macrophage cells and a larger amount of the second biomarker secreted from M2 macrophage cells indicates the proliferative phase of wound healing.
- the first biomarker is calgranulin A/B, TNF-a, MPO, or MMP and the second biomarker is CD204 or CD206.
- the amount of CD204 or CD206 is less than twice the amount of calgranulin A/B, TNF-a, MPO, or MMP, then the wound is in the inflammatory phase of wound healing. Conversely, if the amount of CD204 or CD206 is equal to or greater than twice the amount of calgranulin A/B, TNF-a, MPO, or MMP, then the wound is in the proliferative stage of wound healing.
- the first biomarker is pro-collagen or collagen and the second biomarker is fibronectin.
- the amount of pro-collagen or collagen is greater than the amount of fibronectin, the inflammatory phase of wound healing is indicated. Conversely, if the amount of pro-collagen or collagen is less than the amount of fibronectin, the proliferative phase of wound healing is indicated.
- the present invention provides a method of determining the phase of wound healing comprising: a) detecting the amount of a first biomarker and a second biomarker in a sample from a wound comprising contacting the sample with a solid surface comprising a first agent that is specific to the first biomarker and a second agent that is specific to the second biomarker; and b) comparing the amount of the first biomarker and the amount of the second biomarker, wherein the relative amount of the first biomarker to the second biomarker indicates the phase of wound healing.
- terapéuticaally effective refers to an amount of cells and/or therapeutic composition (such as a therapeutic polynucleotide and/or therapeutic polypeptide) that is employed in methods of the present invention to achieve a therapeutic effect, such as wherein at least one symptom of a condition being treated is at least ameliorated.
- FIG. 1 Schematic diagram of diagnostic detection device for chronic wounds.
- FIG. 2 ELISA analysis of neutrophil- and fibroblast-specific biomarkers.
- FIG. 3 Schematic diagram of digital diagnostic device for rapid detection of status of a wound.
- the disclosed devices and methods provide an easy, rapid, bed-side diagnostic of the wound status. This provides clinicians with a decision tree on "what to do next” by providing information to the healing status of the wound.
- biomarkers from wound fluid, without disrupting the underlying wounded tissue, to accurately diagnose whether a wound is within the normal trajectory of healing or to identify the stage where it has arrested, without causing further damage to the patient.
- diagnostic tests are presented that (1) detect secreted biomarkers and (2) measure the ratio of these markers in a sample. These diagnostic tests will be indicative of whether a wound is stalled in the inflammatory phase or has progressed into the proliferative phase of wound healing without disrupting the underlying damaged tissue.
- the wound healing process is subdivided into three phases: (1) inflammation, (2) proliferation and (3) remodeling.
- the time course of the different cells that appear in the wound during the healing process has been characterized extensively. For example, neutrophils are predominant during inflammation whereas fibroblasts are predominant during the proliferative phase.
- the body mounts an inflammatory response which is characterized by the recruitment of granulocytes, predominantly neutrophils, and cells from the monocyte-macrophage lineage to the site of injury. While neutrophilic inflammation is traditionally regarded as a normal acute response, the continual presence of neutrophils is often associated with delayed wound healing and chronically stalled wounds.
- fibroblasts are the predominant cell-type found during the proliferative phase of a healthy, healing wound.
- the present invention provides a method that contains and detects locally derived biomarkers, which may be used for point-of-care diagnostic purposes (FIG. 1).
- the sample that is analyzed by the devices and methods disclosed herein may be from any desirable source.
- the sample may be collected from a wound exudate.
- the sample may be obtained by aspirating the fluid from the aspirate of a syringe into the wound bed and transferring the contents into a collection vial.
- collection of wound exudates during "negative pressure wound therapy,” “reduced pressure therapy,” or “vacuum therapy” may be used for detection of biomarkers.
- absorbent filter-paper may be placed within a separate device that titrates away a small amount of wound exudates during "negative pressure wound therapy,” “reduced pressure therapy,” or “vacuum therapy.”
- the wound exudate may be collected directly on a wound dressing.
- the sample may be collected from other sources.
- the biomarkers may be extracted from tissue which is either adhered to dressing material (upon removal of the dressing from the wound) or from non-necrotic tissue removed during debridement of the wound. The extraction would possibly entail a soak (lysis) and/or a soak and grind step to release biomarkers.
- the devices and methods disclosed herein utilize biomarkers.
- a biomarker is a measurable biological substance, or signature, that serves as a specific indicator of normal or abnormal biologic processes, often useful for evaluation of pharmacologic responses to therapeutic interventions.
- biomarkers could form the basis for detection of neutrophils and fibroblasts in wounds. Detection of neutrophil-secreted factors and fibroblast-secreted factors may serve as specific biomarkers for the development of a ratio-based diagnostic for determining the phase of wound healing.
- biomarkers are neutrofil- or fibroblast-specific biomarkers.
- biomarkers are secreted from Ml or M2 macrophages.
- Other cell-specific secreted biomarkers may be recognized by a person having ordinary skill in the art.
- the secreted biomarkers may be used in any desirable combination with the disclosed methods and devices.
- the methods and devices may use 1, 2, 3, 4, 5, 10, 15, 20, or more biomarkers.
- the method or device may use one or more neutrophil-secreted factors in combination with one or more fibroblast-secreted factors.
- the method or device may use one or more factors secreted from Ml macrophages in combination with one or more factors secreted from Ml macrophages.
- Other desirable combinations of biomarkers may be recognized by a person having ordinary skill in the art.
- the devices and methods disclosed herein utilize biomarkers that are secreted from neutrophil cells.
- the neutrophil- secreted biomarker is myeloperoxidase (MPO), neutrophil elastase (nElastase), Human Neutrophil Lipolin (HNL), Lactoferrin, Lysozyme, Neutrophil Gelatinase-Associated Lipocalin (NGAL), Human Neutrophil Elastase Anti-Neutrophil Cytoplasmic Antibodies (HNE ANCA's), MMP9, Proteinase 3, Serpin Peptidase Inhibitor Clade B and D, Reactive Oxygen Species (PvOS), or Reactive Nitrogen Species (RNS). 2. Fibroblast-Secreted Factors
- the devices and methods disclosed herein utilize biomarkers that are secreted from fibroblast cells.
- a person having ordinary skill in the art would recognize a variety of secreted fibroblast-specific biomarkers.
- the fibroblast-secreted biomarker is FGF2, FGF10, Fibroblast-specific protein 1 (FSP1), prolyl-4-hydroxylase (5B5), Insulin Growth Factor- 1 (IGF-1), Tetranectin, Collagen alpha 1, 2, and 3 chains, SERPINA1, or Compliment Components.
- Ml macrophages direct an inflammatory response while M2 macrophages initiate repair and angiogenesis.
- M2 macrophages compared to Ml macrophages, produce low amount of tumor necrosis factor-a (TNF-a) and high amounts of other detectable markers. Therefore, distinction between Ml macrophages (that produce pro-inflammatory cytokines) and M2 macrophages (that act to dampen inflammatory responses and promote angiogenesis, tissue remodeling and repair) are also indicative of the phases of wound healing.
- TNF-a tumor necrosis factor-a
- biomarkers specific to Ml or M2 macrophages include, but are not limited to, calgranulin A/B, Cystatin A, SI 00 Calcium Binding Proteins (specific for Ml) and CD 163, CD204, CD206, AM-3K, CSF-1R (colony-stimulating factor-1 receptor), a specific marker of macrophages, EMR1 (epidermal growth factor module-containing mucin-like receptor 1), or F4/80 (specific for M2).
- EMR1 epidermal growth factor module-containing mucin-like receptor 1
- F4/80 specific for M2
- an analysis of levels of pro-collagen or collagen versus fibronectin is also indicative of either the proliferative phase (collagen) or the inflammatory phase (fibronectin) of wound healing.
- the disclosed methods and devices utilize the amount of a biomarker or the ratio between selected biomarkers to determine the phase of wound healing.
- the ratio between a neutrophil-secreted factor and a fibroblast-secreted factor will be monitored.
- an amount of the fibroblast-secreted factor that is greater than the amount of the neutrophil-secreted factor indicates that the wound from which the sample was collected is in the proliferation phase or is properly healing.
- the amount of the fibroblast-secreted factor is greater than the neutrophil- secreted factor by 2-fold or greater, then the wound is in the proliferative stage.
- the ratio of neutrophil-secreted factor to fibroblast-secreted factor is 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, 1:3, 1:4, 1:5, 1:10, or greater or any ratio derivable in between.
- an amount of the neutrophil-secreted factor that is greater than the amount of the fibroblast-secreted factor indicates that the wound from which the sample was collected is in the inflammation phase or is not properly healing.
- the amount of the neutrophil-secreted factor is greater than the fibroblast-secreted factor by 2- fold or greater, then the wound is in the inflammation stage.
- the ratio of fibroblast-secreted factor to neutrophil-secreted factor is 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, 1:3, 1:4, 1:5, 1:10, or greater or any ratio derivable in between.
- the ratio between an Ml -secreted factor and an M2- secreted factor will be monitored.
- an amount of the M2-secreted factor that is greater than the amount of the Ml -secreted factor indicates that the wound from which the sample was collected is in the proliferation phase or is properly healing.
- the amount of the M2-secreted factor is greater than the Ml -secreted factor by 2-fold or greater, then the wound is in the proliferative stage.
- the ratio of Ml- secreted factor to M2-secreted factor is 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, 1:3, 1:4, 1:5, 1:10, or greater or any ratio derivable in between.
- an amount of the Ml -secreted factor that is greater than the amount of the M2-secreted factor indicates that the wound from which the sample was collected is in the inflammation phase or is not properly healing.
- the amount of the Ml -secreted factor is greater than the M2-secreted factor by 2-fold or greater, then the wound is in the inflammation stage.
- the ratio of M2-secreted factor to Ml -secreted factor is 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, 1:3, 1:4, 1:5, 1:10, or greater or any ratio derivable in between.
- biomarkers are secreted from a cell-type specific to the inflammation stage of wound healing and one or more biomarkers are secreted from a cell-type specific to the proliferation stage of wound healing.
- the present invention provides devices that are useful to detect and/or visualize one or more biomarkers from wound fluid. These devices may comprise a surface and at least one agent that is specific to a desired biomarker.
- the surface may be any surface to which the desired agents may be attached, including but not limited to a wound dressing or a microarray.
- the device may be a microfluidic device, e.g., similar to a pregnancy test.
- the device may be in the tubing or tubing connectors of a device designed for "negative pressure wound therapy,” “reduced pressure therapy,” or “vacuum therapy.”
- the tubing may change color when exudates with excessive neutrophil biomarkers or excessive fibroblast markers are present.
- the device may be in the form of a canister or a sticker on the bottom of a canister that turns color.
- the device may be a handheld device that is programmed to detect the levels of these proteins and to digitally display a final reading (FIG. 3).
- the agent specific to the biomarker may be any agent that can bind specifically to the desired biomarker. Examples include, but are not limited to, aptamers, ligands, antibodies, peptide sequences or other binding agents known to those having skill in the art.
- collection of wound exudates during "negative pressure wound therapy,” “reduced pressure therapy,” or “vacuum therapy” may be used for detection of biomarkers.
- the absorbent filter-paper, covalently linked with aptamer, ligand, or antibody may be placed within a separate device that titrates away a small amount of wound exudates during "negative pressure wound therapy,” “reduced pressure therapy,” or “vacuum therapy.”
- the device is coated or bound with agents that bind to the biomarkers.
- the agents may be bound chemically (ionic or covalently) or physically to the wound dressing.
- the agents are bound by chemical grafting, polymer-binder, bi- functional chemical binding (e.g., silane or pluronic), hydrophilic or electrochemical binding, or plasma coating.
- the device may be coated with the agents. 1. Wound Dressings
- the device may be a wound dressing.
- the wound dressing may contain one or more agents specific to a desired biomarker.
- wound dressing used herein is taken to include any pharmaceutically acceptable wound covering or support matrix.
- suitable wound dressing materials include, but are not limited to, a) films, including those of a semipermeable or a semi-occlusive nature such as polyurethane copolymers, polyurethane film, acrylamides, acrylates, paraffin, polysaccharides, cellophane and lanolin; b) hydrocolloids including carboxymethylcellulose protein constituents of gelatin, pectin, and complex polysaccharides including Acacia gum, guar gum and karaya, which may be utilized in the form of a flexible foam, formulated in polyurethane, or formulated as an adhesive mass such as polyisobutylene; c) polymers such as agar, starch or propylene glycol, which typically contain about 80% to about 90%
- the device may be a microarray.
- the microarray may have one or more agents specific to a desired biomarker disposed on its substrate.
- An “array” refers a device consisting of a substrate, typically a solid support having a surface adapted to receive and immobilize a plurality of different protein, peptide, and/or nucleic acid species (i.e., capture or detection reagents) that can used to determine the presence and/or amount of other molecules (i.e., analytes or biomarkers) in biological samples such as wound exudate.
- a “microarray” refers to an array wherein the different detection reagents are disposed on the substrate.
- the agents may be bound chemically (ionic or covalently) or physically to the microarray.
- the detection of the biomarker presence may be performed in any manner known to those having skill in the art.
- the method by which the biomarkers are detected may be related to the type of device used.
- simultaneous detection of several biomarkers may be performed to provide a more accurate determination of the wound status.
- the biomarkers bind to the specific agent on the device.
- the bound biomarkers may be detected by any appropriate method known to those having skill in the art.
- the presence of the secreted biomarkers may be detected using ELISA, immunofluorescence test, microarray, luminescence test, blot, radioimmunoassay, Western blot or dot blot.
- the presence of the secreted biomarkers may be detected using colorimetric detection methods. These methods may result in visual color changes that correlate with the presence or absence of wound healing biomarkers.
- the presence of secreted biomarkers may be detected using Mass Spectrometry, Fourier transform infrared spectroscopy (FTIR), Polymerase Chain Reaction (PCR), Quantitative Real-Time PCR, or Northern Blot.
- FTIR Fourier transform infrared spectroscopy
- PCR Polymerase Chain Reaction
- Quantitative Real-Time PCR or Northern Blot.
- the secreted levels of biomarkers of interest was assess in cultures of distinct cell types to allow one skilled in the art to assess biomarkers that are unique to each cell type and to determine levels of the unique biomarkers.
- the two cell types used were HL60 (Human Promyelocytic Leukemia) cells and HDFa (Adult Human Dermal Fibroblasts).
- the HL60 cells were induced to differentiate into neutrophil-like cells using 1.2% DMSO. Comparable numbers of cells were cultured for comparable amounts of time at 37°C in 5% C0 2.
- Conditioned media (containing secreted factors from each cell type) was assessed for the presence of MPO, FGF2 and other analytes using a traditional ELISA technique (Table 1).
- ELISA Enzyme-linked immunosorbent assay
- an antigen typically a protein
- an unknown amount of antigen, in the test sample is affixed specifically or non-specifically to a surface.
- a specific antibody is then applied over the surface so that it can bind to the test antigen.
- This antibody is typically linked to an enzyme which will be converted into a detectible signal (commonly a color change) upon addition of a chemical substrate.
- FGF Fibroblast Growth Factor
- MPO Myeloperoxidase
- MMP Matrix Metalloproteinase
- TNF Tumor Necrosis Factor
- TGF Transforming Growth Factor
- HL60 Human Pro-myelocytic Leukemia Cell Line
- HDFa Human Dermal Fibroblasts from an adult source
- HEKa Human Epidermal Keratinocyte from an adult source
- N/A not available
- Table 2 shows the concentration range for the analyte that were measured in the discrete cell culture media during the experiment. For example, in HL-60 conditioned media, concentrations of MPO between 6.4 - 16 ng/mL were measured.
- MPO was only detected in conditioned media taken form induced HL60- neutrophil cultures and was completely absent from conditioned media obtained from cultured fibroblasts. This demonstrates that MPO is a good candidate biomarker for the specific detection of neutrophils vs. fibroblasts (FIG. 2).
- HL60 cells were also induced with 0.5% DMSO to attain a neutrophil-like culture. Induced HL60 and HDFa cells were then grown for comparable amounts of time using standard cell culture conditions. Conditioned media was tested for presence of NElastase, FGF- 2, TGF- ⁇ and MPO.
- FGF2 was only detected in conditioned media taken from fibroblasts and this biomarker was, in large part, absent from conditioned media obtained from cultured induced HL60 cells, signifying that this secreted biomarker is suitable for distinguishing fibroblasts from neutrophils in a wound environment.
- Exclusion criteria include: active systemic infection, immunodeficiency disease, anemia (hematocrit ⁇ 26), and/or receiving steroids, immunosuppressive, or cytotoxic medications.
- Test samples are collected first by recording the patients' demographic information (e.g., age of subject, gender, ethnicity, etc), and wound information including: duration of wound, date of future or previous surgeries, type of wound (acute or chronic), and previous wound treatment.
- Test samples are prepared as follows: the wound area is traced and depth is measured in order to calculate the wound area. Based on the wound surface, the amount of saline to be added is calculated and recorded (e.g. , for every 4 cm of wound surface area, add 1 cc of saline).
- a semi-occlusive polyurethane film dressing (Tegaderm, 3M, St. Paul, Minnesota or equivalent) is placed over the wound and wound fluid is collected by piercing the film dressing with a sterile 21 gauge stainless steel needle attached to a syringe and aspirating the contents into the wound bed between the polyurethane film dressing and wound bed itself prior to transfer into a cryo-supported polypropylene tube.
- Analyses for MPO, nElastase, FGF2, and TGFp is performed by ELISA as described above. The ratio between MPO:FGF2 and nElastase:FGF2 found within each wound fluid is determined and is used as diagnostic for the wound. A wound is properly healing if FGF2 > MPO by 2-fold or greater or if FGF2 > nElastase by 2-fold or greater. Levels of FGF2 lower than 2-fold relative to MPO or nElastase is indicative of a "blocked" or non-healing wound.
- compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of some embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
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AU2011332864A AU2011332864A1 (en) | 2010-11-23 | 2011-11-23 | Devices and methods for the diagnosis and treatment of wounds using biomarkers |
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CA2808644A CA2808644A1 (en) | 2010-11-23 | 2011-11-23 | Devices and methods for the diagnosis and treatment of wounds using biomarkers |
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US20170247758A1 (en) * | 2014-08-18 | 2017-08-31 | Drexel University | Methods, computer-readable media, and systems for assessing samples and wounds, predicting whether a wound will heal, and monitoring effectiveness of a treatment |
MX2018011801A (en) | 2016-03-30 | 2019-12-16 | Convatec Technologies Inc | Detecting microbial infections in wounds. |
GB201614053D0 (en) * | 2016-08-17 | 2016-09-28 | Microarray Ltd | Determining the condition of a wound |
WO2018034932A1 (en) | 2016-08-18 | 2018-02-22 | Kci Licensing, Inc. | In-line diagnostic tool for negative-pressure therapy |
EP3312608B1 (en) * | 2016-10-24 | 2019-10-02 | Akribes Biomedical GmbH | Methods for identifying a non-healing skin wound and for monitoring the healing of a skin wound |
WO2018079689A1 (en) * | 2016-10-28 | 2018-05-03 | 公益財団法人がん研究会 | Biomarker, method for searching disease-related gene, and renal cancer marker |
CN109576221B (en) * | 2018-10-12 | 2022-05-27 | 新乡医学院第一附属医院 | Method for preparing neutrophil microvesicles in vitro |
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WO2014114300A1 (en) * | 2013-01-25 | 2014-07-31 | Aarhus Universitet | Novel disease-marker |
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JP2013544361A (en) | 2013-12-12 |
EP2643693A2 (en) | 2013-10-02 |
CN103097892A (en) | 2013-05-08 |
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