DETERMINING THE CONDITION OF A WOUND

Abstract

A product for monitoring the condition of the wound comprising a biologically inert matrix which absorbs wound exudate and one or more reagents on or in the matrix for measuring one or more markers comprised within the wound exudate. A change in the one or more reagents caused by the one or more markers comprised within the wound exudate provides a visual indication of an alteration in the condition of the wound. Companion wound dressings, kits and methods are also provided.

Claims

1. A product for monitoring the condition of a wound comprising: (i) a biologically inert matrix which absorbs wound exudate (ii) one or more reagents on or in the matrix for measuring one or more markers comprised within the wound exudate wherein a change in the one or more reagents caused by the one or more markers comprised within the wound exudate provides a visual indication of an alteration in the condition of the wound.

2. The product according to claim 1 wherein the one or more reagents comprise a complete test unit integrated on or in the matrix.

3. The product according to claim 1 wherein the one or more reagents form a discrete reaction zone on or within the matrix.

4. The product according to claim 1 wherein the alteration is a deterioration.

5. The product according to claim 1 wherein the matrix is able to absorb and retain a volume of wound exudate sufficient for further analysis of the wound exudate.

6. The product according to claim 5 wherein the matrix has the capacity to absorb a volume of at least 0.2 ml wound exudate.

7. The product according to claim 1 wherein the matrix is dimensioned to facilitate positioning between a wound dressing and the wound.

8. The product according to claim 1 wherein the matrix comprises: (i) a first matrix portion comprising one or more reagents on or in the matrix portion for measuring one or more markers comprised within the wound exudate; and (ii) a second matrix portion which is able to absorb and retain a volume of wound exudate sufficient for further analysis of the wound exudate.

9. The product according to claim 8 wherein: (a) the two matrix portions are laminated together; or (b) the two matrix portions are separate, independent components.

10. The product according to claim 8 wherein the surface of the first matrix portion that is not in contact with the second matrix portion is coated or surrounded by a transparent protective layer.

11. The product according to claim 1 wherein the matrix does not measurably alter the condition of the exudate or its components once absorbed.

12. The product according to claim 1 wherein the matrix is sufficiently resistant to compression to allow the matrix to maintain a structure suitable to absorb sufficient volumes of wound exudate for further testing.

13. The product according to claim 1 wherein the matrix is composed of a porous material.

14. The product according to claim 1 wherein the matrix comprises, or is composed of, a material selected from any one or more of: (i) polyurethane; (ii) polyethylene; (iii) cellulose fibres; and/or (iv) porous hydrophilic plastic.

15. The product according to claim 1 wherein, when the product is formed of two matrix portions, the two matrix portions comprise, or are composed of, the same or different materials.

16. The product according to claim 15 wherein the second matrix portion is comprised or composed of polyurethane, optionally polyurethane foam.

17. The product according to claim 14 wherein the polyurethane is a non-isocyanate based polyurethane.

18. The product according to claim 1 wherein: (a) the one or more reagents are insoluble in aqueous conditions; and/or (b) the one or more reagents comprise a cross-linked polymer; and/or (c) the one or more reagents are dried into the matrix; and/or (d) the one or more reagents are conjugated to the matrix.

19. The product according to claim 1 wherein the change in the one or more reagents is degradation of the one or more reagents.

20. The product according to claim 19 wherein degradation of the one or more reagents by one or more markers present in the wound exudate reveals a visible symbol in the matrix, otherwise visually concealed by the one or more reagents.

21. The product according to claim 1 wherein the one or more reagents comprise: (i) a protease substrate; and/or (ii) a myeloperoxidase substrate.

22. The product according to claim 1 wherein: (a) the one or more reagents comprise a substrate for matrix metalloprotease collagenase; and/or (b) the one or more reagents are labelled; and/or (c) the one or more reagents are coloured.

23. The product according to claim 1 wherein the one or more reagents are dried with: (i) activated carbon particles; and/or (ii) coloured micro-particles; entrained within the dried reagent mass.

24. The product according to claim 23 wherein the micro-particles comprise: (i) copper phthalocyanine tetrasulfonic acid tetrasodium salt; (ii) latex microspheres; and/or (iii) polystyrene microspheres.

25. The product according to claim 22 wherein: (a) the one or more reagents are labelled with a fluorescent label that is quenched unless and until the one or more reagents are changed, for instance by cleavage, by one or more markers present in the wound exudate; and/or (b) the one or more reagents are chemically conjugated to one or more dye molecules.

26. The product according to claim 1 wherein the one or more reagents comprise collagen, optionally gelatin.

27. The product according to claim 1 wherein: (a) the one or more reagents comprise a substrate for a serine protease, optionally a neutrophil elastase, in particular a human neutrophil elastase; and/or (b) the one or more reagents comprise elastin; and/or (c) the one or more reagents comprise a substrate for a cathepsin protease, optionally cathepsin G; and/or (d) the one or more reagents comprise a substrate for papain-family enzymes, such as staphopain from Staphylococcus aureus.

28. The product according to claim 1 wherein: (a) modification of the one or more reagents by exudate from the wound indicates the need for further analysis of the exudate; and/or (b) the absorbed exudate can be retrieved from the matrix for further analysis; and/or (c) the change in the one or more reagents only occurs if the one or more markers are present in the wound exudate at or above a pre-determined threshold level.

29. A wound dressing comprising the product according to claim 1.

30. A kit comprising the product according to claim 1 and a vessel suitable for safe containment and shipping of the product.

31. A kit according to claim 30 wherein, when the matrix comprises two portions, the two portions are provided as two separate components, optionally wherein the first and second matrix portions can be connected to one another.

32. A method for monitoring the condition of a wound on a subject comprising: (a) placing a product according to claim 1 in contact with the wound under a wound dressing; (b) leaving the product in contact with the wound for a pre-determined amount of time; (c) determining the presence or absence of a visual indication of an alteration in the condition of the wound by the product; wherein the presence of the visual indication signals the need for further analysis of the wound exudate.

33. The method according to claim 32 wherein, when the product comprises a matrix comprising two portions and the two portions are separate components not connected to each other, each portion is independently placed in contact with the wound for a pre-determined amount of time.

34. The method according to claim 32 wherein, when the product comprises a matrix comprising two portions and the two portions are separate components, prior to, or as part of, step (a) the first and second matrix portions are connected to one another.

35. The method according to claim 32 wherein the visual indication is combined with one or more indications selected from: (i) the smell of the wound (ii) the total volume of exudate (iii) the appearance of the wound (iv) the systemic condition of the subject in order to determine the need for further analysis of the wound exudate.

36. The method according to claim 32 wherein the method further comprises: (d) removal of the product from contact with the wound; (e) retrieving the exudate absorbed by the product; (f) analysing the retrieved exudate in order to determine the condition of the wound.

37. The method according to claim 32 wherein step (d) further comprises storage of the product in a vessel suitable for safe containment and shipping prior to steps (e) and (f).

38. The method according to claim 32 wherein the method is repeated at intervals in order to facilitate longitudinal monitoring of the condition of the wound, optionally wherein the intervals are daily, weekly or monthly or a combination thereof.

39. The method according to claim 32 wherein the absence of any visual indication of an alteration in the condition of the wound indicates that existing treatment of the wound should be continued.

40. The method according to claim 32 wherein the wound is a chronic wound.

41. A product according to claim 1 for use in a method comprising: (a) placing the product in contact with the wound under a wound dressing; (b) leaving the product in contact with the wound for a pre-determined amount of time; (c) determining the presence or absence of a visual indication of an alteration in the condition of the wound by the product; wherein the presence of the visual indication signals the need for further analysis of the wound exudate.

Description

DESCRIPTION OF THE FIGURES

[0309] FIG. 1A is a schematic illustrating one embodiment of the invention wherein a product as described herein is placed in contact with a wound underneath a wound dressing.

[0310] FIG. 1B is a schematic illustrating the generation of a visual indication by a product as described herein following absorption of the wound exudate and modification of the one or more reagents on or in the matrix by one or more markers present in the wound exudate.

[0311] FIG. 1C is a schematic illustrating one embodiment of the invention wherein a product as described herein is placed in contact with a wound underneath a wound dressing wherein the product comprises a matrix comprising a first and second portion.

[0312] FIG. 1D is a schematic illustrating the generation of a visual indication by a product as described herein comprising a matrix comprising a first and second portion following absorption of the wound exudate and modification of the one or more reagents on or in the first matrix portion by one or more markers present in the wound exudate.

[0313] FIG. 2A is a schematic illustrating a further embodiment of the invention wherein a product as described herein further comprises a reaction vessel.

[0314] FIG. 2B is a schematic illustrating the generation of a visual indication by a product as described herein comprising a reaction vessel following absorption of the wound exudate and modification of the one or more reagents on or in the matrix and contained in the reaction vessel by one or more markers present in the wound exudate.

[0315] FIG. 3 provides a flowchart to illustrate one embodiment of the methods of the invention wherein a product as described herein provides a visual indication of an alteration of a change in the condition of a wound.

[0316] FIG. 4 provides a flowchart to illustrate a further embodiment of the methods of the invention wherein the absence of a visual indication by a product as described herein indicates there has been no change in the condition of a wound.

[0317] FIG. 5 demonstrates one embodiment of the invention able to detect active papain in a sample using gelatin infiltrated with copper phthalocyanine tetrasulfonic acid tetrasodium salt (CPSS) to form a gelatin-CPSS complex.

[0318] FIG. 6 demonstrates one embodiment of the invention able to detect active human neutrophil elastase in a sample using gelatin infiltrated with copper phthalocyanine tetrasulfonic acid tetrasodium salt (CPSS) to form a gelatin-CPSS complex.

[0319] FIG. 7 demonstrates one embodiment of the invention able to detect active matrix metallopeptidase 9 in a sample using gelatin infiltrated with copper phthalocyanine tetrasulfonic acid tetrasodium salt (CPSS) to form a gelatin-CPSS complex.

[0320] FIGS. 8A-C demonstrate one embodiment of the invention able to detect active papain in a sample using gelatin infiltrated with dyed polystyrene microspheres (PSM) to form a gelatin-PSM complex.

[0321] FIGS. 9A-B demonstrate one embodiment of the invention able to detect active matrix metallopeptidase 9 in a sample using gelatin infiltrated with dyed polystyrene microspheres (PSM) to form a gelatin-PSM complex.

[0322] FIGS. 10A-B demonstrate one embodiment of the invention able to detect active human neutrophil elastase in a sample using gelatin infiltrated with dyed polystyrene microspheres (PSM) to form a gelatin-PSM complex.

DETAILED DESCRIPTION OF THE INVENTION

[0323] The invention will now be described, without limitation but solely to aid understanding of the invention, by reference to the FIGS.

[0324] A product (1) as described herein is shown schematically in FIG. 1A. The product (1) comprises a biologically inert matrix (2) which can absorb wound exudate and one or more reagents (3), in this case activated carbon-labelled collagen, on or in the matrix (2). The one or more reagents (3) may be dried into, or conjugated to, the matrix (2) and, in the case of activated carbon-labelled collagen, make the whole or a substantial part of the matrix appear black in colour, forming a test unit or reaction zone. The one or more reagents (3) are used to measure one or more components (markers) comprised within exudate released by a wound. The one or more reagents (3) can be specifically modified by one or more markers of the wound exudate if such markers are present, or present at or above a threshold concentration in the exudate.

[0325] When the product (1) is placed in contact with the wound (4), which may be a chronic wound, on a subject (5) underneath a wound dressing (6), the matrix (2) absorbs wound exudate. If the wound exudate comprises one or more markers capable of specifically modifying the one or more reagents this causes a modification in the one or more reagents that consequently provides a visual indication of an alteration in the condition of the wound, such as a deterioration of the wound. In this case, the activated carbon-labelled collagen (3) on or in the matrix (2) is degraded into fragments by collagenase enzymes present in the wound exudate, in particular if the collagenase enzymes are present at or above a threshold level. Once degraded, the fragments of activated carbon-labelled collagen are free to dissociate from the matrix. In this case, as shown in FIG. 1B, dissociation of the activated carbon-labelled collagen fragments from the matrix removes the black colouration that was attributed to the reaction zone of the matrix by the activated carbon label and reveals a visual symbol printed on the product such as a cross (7). The revelation of the visual symbol indicates to the caregiver, such as a district nurse or family member, at the time of re-dressing the wound and replacement of the product (1) with a new, sterile product, that there exists a need for further downstream analysis of the wound exudate such as laboratory testing.

[0326] An alternative embodiment is shown in FIGS. 1C and 1D. In this embodiment, the product (1) comprises a first matrix portion (8) and a second matrix portion (9), connected to one another at a connecting surface (10). Both portions are biologically inert and able to absorb wound exudate. The first matrix portion (8) comprises one or more reagents (11), in this case gelatin entrained with coloured polystyrene microspheres (PSM), on or in the first matrix portion (8). The gelatin-PSM complex makes the whole or a substantial part of the first matrix portion appear black in colour, forming a test unit or reaction zone. The one or more reagents (11) are used to measure one or more components (markers) comprised within exudate released by a wound. The one or more reagents (11) can be specifically modified by one or more markers of the wound exudate if such markers are present, or present at or above a threshold concentration in the exudate.

[0327] When the product (1) is placed in contact with the wound (4), which may be a chronic wound, on a subject (5) underneath a wound dressing (6), the first and second matrix portions, (8) and (9) respectively, absorb wound exudate. Typically, wound exudate is absorbed by the first matrix portion (8) via fluid connection with the second matrix portion (9) at the connecting surface (10). If the wound exudate comprises one or more markers capable of specifically modifying the one or more reagents this causes a modification in the one or more reagents that consequently provides a visual indication of an alteration in the condition of the wound, such as a deterioration of the wound. In this case, the gelatin of the gelatin-PSM complex (11) entrained within the first matrix portion (8) is degraded into fragments by gelatinase enzymes present in the wound exudate, in particular if the gelatinase enzymes are present at or above a threshold level. Once the gelatin is degraded, the PSM is free to disperse throughout and/or dissociate from the matrix. In this case, as shown in FIG. 1 D, dispersal and/or dissociation of PSM from the matrix dissipates the colouration that was attributed to the reaction zone of the matrix by the gelatin-PSM complex and reveals a visual symbol printed on the product such as a cross (7). The revelation of the visual symbol indicates to the caregiver, such as a district nurse or family member, at the time of re-dressing the wound and replacement of the product (1) with a new, sterile product, that there exists a need for further downstream analysis of the wound exudate such as laboratory testing.

[0328] In other embodiments, only the first matrix portion (8) is placed in contact with the wound, thus, the product functions solely as an in-wound protease activity detector. These embodiments are advantageous as they are extremely simple to operate and interpret. In further embodiments, the first matrix portion (8) and second matrix portion (9) are not connected to each other but instead are placed independently in the wound. The first matrix portion (8) provides a visual indication of an alteration in the condition of the wound, such as a deterioration of the wound, as described above. The second matrix portion (9) absorbs wound exudate in an amount sufficient for downstream analysis of the wound exudate.

[0329] While the cross (7) provides a positive test result, and is therefore advantageous, it is not essential. Instead dissipation of the colouration can be used as an outcome of the test without revealing a further symbol.

[0330] As shown in FIG. 2A, in certain embodiments, the product (21) further comprises a reaction vessel (27) extending from the matrix (22) and which is in fluid connection (28) with the matrix (22), such connection may be via one or more capillary flow paths. The reaction vessel (27) extends sufficiently from the matrix (22) to be positioned at least in part outside of a wound dressing (26) applied to the wound (24) such that it is visible to a subject. The reaction vessel (27) is exposed to and/or may absorb wound exudate by virtue of the fluid connection (28) with the matrix (22) following absorption of wound exudate via the matrix (22). In certain embodiments, the reaction vessel contains one or more reagents (29) able to specifically detect and measure a one or more markers that may be present within the wound exudate. The one or more reagents (29) contained within the reaction vessel (27) may be the same or different to the one or more reagents (23) on or in the matrix (22). In this case, the one or more reagents (23) on or in the matrix (22) and the one or more reagents (29) contained within the reaction vessel (27) is activated carbon-labelled collagen. In other embodiments, the reaction vessel (29) and reagents therein can replace the reagents in the reaction zone of the matrix (23).

[0331] In this case, the activated carbon-labelled collagen (23) on or in the matrix (22) is degraded into fragments by collagenase enzymes present in the wound exudate. In addition, exposure of the activated carbon-labelled collagen (29) contained within the reaction vessel (27) to collagenase enzymes present in the wound exudate by virtue of the fluid connection (28) with the matrix (22) degrades the activated carbon-labelled collagen (29) into fragments also. Once degraded, the fragments of activated carbon-labelled collagen are free to dissociate from the reaction vessel and matrix. In this case, as shown in FIG. 2B, dissociation of the activated carbon-labelled collagen fragments from the reaction vessel (27) and matrix (22) removes the black colouration that was attributed to the reaction vessel (27) and matrix (22) by the activated carbon label and reveals a visual symbol, such as a cross, printed on the reaction vessel (211) and matrix (210).

[0332] As a consequence of the reaction vessel (27) extending sufficiently from the matrix (22) to be positioned outside of a wound dressing (26), the subject suffering from the wound or the caregiver at the point of care can observe the visual symbol, such as a cross, printed on the reaction vessel (211) without needing to remove the wound dressing. As a result, the subject and/or caregiver is able to receive earlier warning of a change in the condition of the wound, such as deterioration, and can therefore seek clinical intervention more quickly. The inclusion of two detectable reactions provides an internal cross-check.

[0333] The presence of the cross in the matrix (210) but not in the reaction vessel (211) may indicate that excess collagenase activity is present but that the volume of exudate absorbed was not sufficient to saturate the reaction vessel (27).

[0334] In addition, the reaction vessel (27) provides a handle means by which the product (21) can be removed from the wound (24) without requiring contact with the matrix (22) thereby decreasing the possibility of contamination of the wound exudate absorbed by the matrix (22) via said contact. In some embodiments, the reaction vessel can be cleaved from the matrix (22), for instance at or near to the fluid connection aperture (28), thereby further minimising the possibility of contamination of the wound exudate absorbed by the matrix (22).

[0335] FIG. 3 provides a flowchart to illustrate one embodiment of the methods of the invention. More specifically, a product as described herein, which may, in certain embodiments, be the product as described above in relation to FIG. 1, is placed in contact with a wound on a subject, underneath a wound dressing, for a pre-determined period of time (31). This period of time allows the product to absorb wound exudate.

[0336] At the end of this period and at the point of care, the caregiver, such as a district nurse or a family member, removes the wound dressing and assesses the product for the presence of a visual indication by the product as a consequence of a modification in the one or more reagents on or in the product matrix caused by an interaction with one or more markers in the wound exudate (32). For instance, in relation to FIGS. 1A-B, the visual indication may be a symbol such as a cross (7) following degradation of the activated carbon-labelled collagen marker on or in the matrix (2) by collagenase present in the wound exudate that has been absorbed by said matrix, optionally wherein the collagenase enzymes are present at or above a threshold level. Alternatively, in relation to FIGS. 1C-D, the visual indication may be a symbol such as a cross (7) following degradation of the gelatin-PSM complex on or in the matrix (2) by gelatinase present in the wound exudate that has been absorbed by said matrix, optionally wherein the gelatinase enzymes are present at or above a threshold level.

[0337] Observance of the visual indication, such as a cross (7), by the caregiver indicates that there has been an alteration in the condition of the wound, which may be deterioration, and that further analysis of the wound exudate is required.

[0338] For those embodiments, as described above in relation to FIG. 2, wherein the product (21) further comprises a reaction vessel (27) extending sufficiently from the matrix (22) to be positioned, at least partially, outside of a wound dressing (26) and which is in fluid connection (28) with the matrix (22), the subject suffering from the wound or the caregiver at the point of care can observe the visual symbol, such as a cross, printed on the reaction vessel (211) without needing to remove the wound dressing. For instance, the visual indication, such as a cross (211), provided by the reaction vessel (27) can alert the subject to a change in the condition of the wound prior to any scheduled appointment with a caregiver and/or clinician, thus, enabling the subject to seek earlier clinical intervention and to alert the caregiver and/or clinician that the condition of the wound has altered.

[0339] The assessment of the visual indication may be combined with one or more other indications including the smell of the wound, the total volume of wound exudate, the appearance of the wound and/or the systemic condition of the subject in order to determine the need for further analysis of the wound exudate. Thus, the absence of a visual indication by the product in the presence of one or more other indications may still ultimately result in a determination that further analysis of the wound exudate is required.

[0340] If further analysis of the wound exudate is required, the product is removed from contact with the wound (33) and placed in a vessel suitable for safe containment and shipping of the product to a laboratory (34). For embodiments where the product comprises a matrix comprising a first and second portion, the second matrix portion and optionally the first matrix portion are placed in a vessel suitable for safe containment and shipping of the product to a laboratory (34). In certain embodiments, such a vessel may comprise a biologically inert internal surface which may not measurably alter the condition of the wound exudate or its components that have been absorbed by the product matrix whilst it was in contact with the wound. In order to minimise contamination of the wound exudate absorbed by the matrix, embodiments of the product comprising a reaction vessel can use said vessel as a handle means to remove the product from contact with the wound. In some embodiments, the reaction vessel can be cleaved from the matrix, for instance at or near to the point of fluid connection with the matrix, so that only the matrix containing the absorbed wound exudate is captured within the containment vessel, thereby further minimising the possibility of contamination of the wound exudate absorbed by the matrix.

[0341] The vessel containing the product which itself contains exudate absorbed from the wound is transported to the laboratory where the product may be released from the vessel and the absorbed wound exudate retrieved from the product (35). In some embodiments, the vessel containing the product which itself contains exudate absorbed from the wound is suitable for and is stored in the laboratory under suitable conditions, for instance at 80 C., to allow retrieval and analysis of the wound exudate at a later point in time.

[0342] Once the wound exudate is retrieved from the product, the product may be disposed of and the wound exudate analysed to determine the condition of the wound (36).

[0343] The process of FIG. 3 may be repeated at intervals in order to facilitate longitudinal monitoring of the condition of the wound. Thus, for instance, following removal of a product which has been in contact with the wound for a pre-determined period of time and which has absorbed wound exudate (33) and prior to re-dressing the wound, a new, sterile product as described herein is placed in contact with the wound underneath the new wound dressing and the process of FIG. 3 repeated. Said intervals may be every 1, 2, 3, 4, 5 or 6 days, weekly or monthly or a combination thereof and may be varied from time to time.

[0344] FIG. 4 provides a flowchart to illustrate an alternative outcome in relation to the visual indication and represents particular embodiments of the invention. In this case, a product as described herein is placed in contact with a wound on a subject, underneath a wound dressing, for a pre-determined period of time (41) (the pre-determined contact time). This period of time allows the product to absorb wound exudate.

[0345] At the end of this period, the product is assessed for the presence of a visual indication by the product as a consequence of a modification in the one or more reagents comprised on or in the product matrix (or contained within the reaction vessel if present) caused by an interaction with one or more markers present in the wound exudate (42).

[0346] If no visual indication is presented by the product, the caregiver, which may be a district nurse or a family member, at the point of care may still remove the product from contact with the wound and send the product away for laboratory analysis of the wound exudate (as illustrated (45)-(48) and as described above) if a pre-determined period of time has passed since exudate from the wound has been sampled (the pre-determined sampling time), in this case a period of greater than or equal to 4 weeks (43). In alternative embodiments, this period may be every 1, 2, 3, 4, 5 or 6 days, weekly or monthly or a combination thereof. Sampling in this manner facilitates longitudinal monitoring of the condition of the wound. If the pre-determined sampling time has passed and the caregiver removes the product from contact with the wound in order to transport the product to the laboratory for testing of the absorbed wound exudate, the caregiver at the point of care replaces it with a new, sterile product, covers with a fresh wound dressing and monitoring of the wound repeats per FIG. 3 or FIG. 4 as appropriate depending on the presence or absence of a visual indication by the product after the pre-determined contact time. It should be noted that the pre-determined sampling time could be expressed in terms of number of products utilised since the last product was sent for testing. For example, every 5.sup.th dressing change the product could be sent for futher testing as described even if no visual indication is presented.

[0347] Alternatively, if no visual indication is presented by the product and the pre-determined sampling time has not passed, the caregiver at the point of care removes the product from contact with the wound, replaces it with a new, sterile product, covers with a fresh wound dressing (44) and monitoring of the wound repeats per FIG. 3 or FIG. 4 as appropriate depending on the presence or absence of a visual indication by the product after the pre-determined contact time.

[0348] The aggregation of data pertaining to the condition of the wound over time via sampling and analysing the wound exudate over time as described in FIGS. 3 and 4 better enables the clinician to understand the progress of the condition of the wound and efficacy of treatment(s). For instance, longitudinal monitoring of the wound exudate as described may indicate to the clinician, in a more rapid and/or quantitative fashion than current procedures, that the condition of the wound is deteriorating over time and thus the present treatment is ineffective. Consequently, the clinician can more rapidly and in a more informed fashion select alternative treatments in order to promote healing of the wound. Alternatively, the data may indicate to the clinician that further tests of the wound exudate and/or wound environment are needed. Furthermore, the aggregated data allows the clinician to develop a visiting schedule in relation to further sampling of the wound exudate and re-dressing of the wound by a caregiver, such as a district nurse or family member, depending on the degree and direction of change in the condition of the wound over time.

[0349] Experimental Section

EXAMPLE 1

[0350] Overview

[0351] Gelatin was mixed with Copper Phthalocyanine Tetrasulfonic Acid Tetrasodium salt (CPSS) to form a gelatin-CPSS complex. The gelatin-CPSS sample was dried down onto a support membrane. To initiate the detection of protease activity, the dried gelatin-CPSS sample was wetted with activated protease solution and incubated at room temperature (typically around 21 C.). If zero protease activity was present, the gelatin-CPSS sample remained intact. If protease activity was present, the gelatin was hydrolysed into smaller, mobile fragments, releasing the embedded CPSS which diffused away from the original site of application. This attenuation and dispersal in colour indicated a positive protease reaction.

[0352] Experimental Protocol

[0353] A 9.1% w/w gelatin (Type A, porcine origin, Sigma G2500) solution in deionised water (DI H.sub.2O) was prepared by adding 1.25 g gelatin powder to 12.5 ml DI H.sub.2O to give a total weight of 13.75 g. The powder was allowed to wet and swell for 5 mins at room temperature (RT) before heating to a minimum of 50 C. The sample was mixed to dissolve the gelatin. 1884 L of glycerol (Sigma G5516) was added to a final concentration of around 1.2% and thoroughly mixed. The sample was kept at a minimum temperature of 40 C., to ensure the gelatin remained in a liquid state. CPSS (Sigma 274011) powder was added to the liquid gelatin mixture to a final concentration of 2 mg/ml. The sample was mixed to dissolve the CPSS. All CPSS dye was adsorbed by the gelatin and no further processing of the gelatin-CPSS mixture was performed. 2 L of the mixture was dropped onto a supportive membrane using a calibrated micro-volume pipette (0.5-3 L). The drop was dried either by air-drying at RT, accelerated using a 37 C. incubator or a drying tunnel at 50 C.

[0354] The action of protease enzyme was then evaluated. Examples of proteases used were papain (DMV, around 1000 u/g), human neutrophil elastase (HNE, Lee Biosolutions (code 342-40)) and matrix metalloprotease 9 (MMP9, Alere SD (special commission)). Papain powder was dissolved into DI H.sub.2O at 1 mg/ml, before dilution into activation buffer (1.7 mM EDTA, 10 mM cysteine-HCl, 200 mM sodium chloride, pH 7) to give the required final working concentrations. Stock HNE enzyme was diluted into activation buffer (50 mM Tris, 10 mM calcium chloride dihydrate, 100 mM sodium chloride, 50 M zinc chloride, 0.025% w/w Brij 35, 0.05% w/w sodium azide, pH 7.4) to give the required final working concentrations. Stock MMP9 enzyme was diluted into activation buffer (50 mM Tris, 10 mM calcium chloride dihydrate, 100 mM sodium chloride, 50 M zinc chloride, 0.025% w/w Brij 35, 0.05% w/w sodium azide, pH 7.4) to give the required final working concentrations.

[0355] Samples of the dry gelatin-CPSS on the support membrane were taken. The membrane was wetted with the specific protease sample until saturation. The samples were left to incubate at RT. At various time points, the integrity of the coloured gelatin-CPSS was examined. A positive result for the presence of protease enzyme was shown by the attenuation of colour due to the digestion of the gelatin and the diffusion of the CPSS molecules away from the site of application. A negative result (i.e. zero active protease) was seen by the original dried gelatin-CPSS indicator remaining in place.

[0356] Results

[0357] Papain

[0358] The results in relation to detecting papain activity are shown in FIG. 5.

[0359] After 72 hours, papain activity was detected using papain concentrations of 0.1, 0.05 and 0.025 mg/ml. The CPSS molecules diffused throughout the supportive disk resulting in an attenuation of the blue colour and confirming protease activity detection. In the absence of active papain (0 mg/mL), the gelatin-CPSS complex remained intact and no attenuation or dispersal of colour was observed.

[0360] HNE

[0361] The results in relation to detecting HNE activity are shown in FIG. 6.

[0362] After 72 hours, HNE activity was detected using HNE concentrations of 0.01 and 0.001 mg/ml. The CPSS molecules diffused throughout the supportive disk resulting in an attenuation of the blue colour and confirming protease activity detection. In the absence of active HNE (0 mg/mL), the gelatin-CPSS complex remained intact and no attenuation or dispersal of colour was observed.

[0363] MMP9

[0364] The results in relation to detecting MMP9 activity are shown in FIG. 7.

[0365] After 72 hours, MMP9 activity was detected using MMP9 concentrations of 0.01 and 0.001mg/ml. The CPSS molecules diffused throughout the supportive disk resulting in an attenuation of the blue colour and confirming protease activity detection. In the absence of active MMP9 (0 mg/mL), the gelatin-CPSS complex remained intact and no attenuation or dispersal of colour was observed.

[0366] Summary

[0367] The above examples show the presence of protease activity by the attenuation or loss of the applied coloured spot. This visual change can be interpreted as a negative-read result, due to the loss of colour. The same visual change can also be interpreted as a positive-read result, by the inclusion of an additional permanent coloured mark, either under or above the coloured gelatin-CPSS which acts to hide the mark. When the gelatin-CPSS has been digested and diffused throughout the supportive pad, the masked coloured mark is thus revealed. The revealing of the mark, or message can be interpreted as a positive result confirming the presence of protease activity.

EXAMPLE 2

[0368] Overview

[0369] Gelatin was mixed with dyed polystyrene microspheres (PSM) to form a gelatin-PSM complex. The gelatin-PSM sample was dried down onto a support membrane. To initiate the detection of protease activity, the dried gelatin-PSM sample was wetted with activated protease solution and incubated at room temperature (typically around 21 C.). If zero protease activity was present, the gelatin-PSM sample remained intact. If protease activity was present, the gelatin was hydrolysed into smaller, mobile fragments, releasing the embedded PSM which diffused away from the original site of application. This attenuation and dispersal in colour indicated a positive protease reaction.

[0370] Experimental Protocol

[0371] A 9.1% w/w gelatin (Type A, porcine origin, Sigma G2500) solution in deionised water (DI H.sub.2O) was prepared by adding 1.25 g gelatin powder to 12.5 ml DI H.sub.2O to give a total weight of 13.75 g. The powder was allowed to wet and swell for 5 mins at room temperature (RT) before heating to a minimum of 50 C. The sample was mixed to dissolve the gelatin. 188 L of glycerol (Sigma G5516) was added to a final concentration of around 1.2% and thoroughly mixed. The sample was kept at a minimum temperature of 40 C., to ensure the gelatin remained in a liquid state. Polystyrene microspheres (PSM, 5% solids, 528 nm diameter (blue) or 10% solids, 240 nm (dark blue)) were added to a gelatin solution (3 L PSM+47 L gelatin) and mixed to give either 0.3% or 0.6% solids final concentration. 2 L of the mixture was dropped onto a supportive membrane using a calibrated micro-volume pipette (0.5-3 L). The drop was dried either by air-drying at RT, or accelerated using a 37 C, incubator or drying tunnel. Protease solutions were prepared as described in Example 5.

[0372] Samples of the dry gelatin-PSM on the support membrane were taken. The membrane was wetted with the specific protease sample until saturation. The samples were left to incubate at RT. At various time points, the integrity of the coloured gelatin-PSM was examined. A positive result for the presence of protease enzyme was shown by the attenuation of colour due to the digestion of the gelatin and the diffusion of the PSM molecules away from the site of application. A negative result (i.e. zero active protease) was seen by the original dried gelatin-PSM indicator remaining in place.

[0373] Results

[0374] Papain

[0375] The results in relation to detecting papain activity are shown in FIGS. 8A-C.

[0376] After only 24 hours, papain activity was detected using papain concentrations of 0.1 and 0.01 mg/ml. The PSM molecules diffused throughout the supportive disk resulting in an attenuation of the blue colour and confirming protease activity detection. In the absence of active papain (0 mg/mL), the gelatin-PSM complex remained intact and no attenuation or dispersal of colour was observed.

[0377] MMP9

[0378] The results in relation to detecting MMP9 activity are shown in FIGS. 9A-B.

[0379] After 72 hours, MMP9 activity was detected using MMP9 concentrations of 0.01, 0.001 and 0.0001 mg/ml. The PSM molecules diffused throughout the supportive disk resulting in an attenuation of the blue colour and confirming protease activity detection. In the absence of active MMP9(0 mg/mL), the gelatin-PSM complex remained intact and no attenuation or dispersal of colour was observed.

[0380] HNE

[0381] The results in relation to detecting HNE activity are shown in FIGS. 10A-B.

[0382] After 72 hours, HNE activity was detected using HNE concentrations of 0.01 and 0.001 mg/ml. The PSM molecules diffused throughout the supportive disk resulting in an attenuation of the blue colour and confirming protease activity detection. In the absence of active HNE (0 mg/mL), the gelatin-PSM complex remained intact and no attenuation or dispersal of colour was observed.

[0383] Summary

[0384] The above examples show the presence of protease activity by the attenuation or loss of the applied coloured spot. This visual change can be interpreted as a negative-read result, due to the loss of colour. The same visual change can also be interpreted as a positive-read result, by the inclusion of an additional permanent coloured mark, either under or above the coloured gelatin-PSM which acts to hide the mark. When the gelatin-PSM has been digested and diffused throughout the supportive pad, the masked coloured mark is thus revealed. The revealing of the mark, or message can be interpreted as a positive result confirming the presence of protease activity.

[0385] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. Moreover, all aspects and embodiments of the invention described herein are considered to be broadly applicable and combinable with any and all other consistent embodiments, including those taken from other aspects of the invention (including in isolation) as appropriate. Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.