Evaluation of wound fluid biomarkers to determine healing in adults with venous leg ulcers: A prospective study

Abstract

Clinical practice guidelines recommend using repeated wound surface area measurements to determine if a chronic ulcer is healing. This results in delays in determining the healing status. This study aimed to evaluate whether any of a panel of biomarkers can determine the healing status of chronic venous leg ulcers. Forty-two patients with chronic venous leg ulcers had their wound measured and wound fluid collected at weekly time points for 13 weeks. Wound fluid was analyzed using multiplex enzymelinked immunosorbent assay to determine the concentration of biomarkers in the wound fluid at each weekly time point. Healing status was determined by examining the change in wound size at the previous and subsequent weeks. Predictive accuracy with 95% confidence intervals (CI) is reported. Of 42 patients, 105 evaluable weekly time points were obtained, with 32 classified as healing, 27 as nonhealing, and 46 as indeterminate. Thirteen biomarkers significantly differed between healing and nonhealing wounds (p < 0.1) and were included in a multivariate logistic regression model. Granulocyte macrophage-colony stimulating factor (p < 0.001) and matrix metalloprotease-13 (p = 0.004) were the best predictors of wound healing. Receiver operating characteristic curves indicated 92% accuracy (95% CI: 85%,100%) for granulocyte macrophagecolony stimulating factor, and 78% accuracy (95% CI: 65%,90%) for matrix metalloprotease-13 in discriminating between healing and nonhealing wounds. This study found that two biomarkers from wound fluid can predict healing status in chronic venous leg ulcers. These findings may lead to the ability to determine the future trajectory of a wound and the ability to modify treatment accordingly. METHODS Study designThis was a prospective cohort study where wound surface area measurements and wound fluid was collected from VLU patients for 13 weeks, or until the wound was healed. The study was performed in compliance with the Declaration of Helsinki Wound Rep Reg (2019) 27 509-518

A prospective clinical trial evaluating changes in the wound microenvironment in patients with chronic venous leg ulcers treated with a hypothermically stored amniotic membrane

“…While this is the first study to investigate the proteomic changes within a VLU treated with a placental‐derived product, this is not the first study to investigate changes in wound fluid of healing and non‐healing chronic wounds. Stacey et al also reported a number of differentially expressed growth factors and cytokines in the wound fluid of healing and non‐healing VLUs including Eotaxin, GM‐CSF, ICAM‐1, IL‐6, IL‐16, MCP‐1, MIP‐1a, MMP‐13, PDGF‐BB, and TIMP‐4 37 . Similarly, Edsberg et al conducted a proteomic analysis on the wound fluid of pressure ulcers and identified 20 proteins which were also differentially expressed between chronic and healed wounds including GM‐CSF, I‐309, INF‐γ IL‐11, IL‐12p40, IL‐15, IL‐1a, IL‐1b, IL‐8, TIMP‐1, TIMP‐2 TNF RI, TNF RII, ICAM‐1, IL‐16, MIP‐1d, MMP‐10, MMP‐13, MMP‐3, and Eotaxin‐2 38 .…”

“…Similarly, Edsberg et al conducted a proteomic analysis on the wound fluid of pressure ulcers and identified 20 proteins which were also differentially expressed between chronic and healed wounds including GM‐CSF, I‐309, INF‐γ IL‐11, IL‐12p40, IL‐15, IL‐1a, IL‐1b, IL‐8, TIMP‐1, TIMP‐2 TNF RI, TNF RII, ICAM‐1, IL‐16, MIP‐1d, MMP‐10, MMP‐13, MMP‐3, and Eotaxin‐2 38 . In this study, we observed similar trends as seen in other studies with TIMP‐4, IL‐1a, and MMP‐10 37,38 . Prior studies have identified that protease levels within the wound bed of VLUs may be predictive of the outcome of the wound, specifically MMP‐1, 39 MMP‐2, 39,40 MMP‐7, 39 and MMP‐9 33,39‐41 ; however, it should be noted that there is no current consensus that protease activity is an indicator of healing in VLUs 42 .…”

“…VLUs in these studies had a mean size of 4.6 to 7.6 cm 2 and a duration of 41.9 to 55.2 weeks, which is generally less severe than ulcers evaluated in our study which had an average size of 8.6 ± 7.9 cm 2 and a 38 In this study, we observed similar trends as seen in other studies with TIMP-4, IL-1a, and MMP-10. 37,38 Prior studies have identified that protease levels within the wound bed of VLUs may be predictive of the outcome of the wound, specifically MMP-1, 39 MMP-2, 39,40 MMP-7, 39 and MMP-9 33,[39][40][41] ; however, it should be noted that there is no current consensus that protease activity is an indicator of healing in VLUs. 42 In our study, we observed that MMP-7 was elevated in VLUs on a healing trajectory relative to nonhealed, VLUs which is consistent with the results reported by Raffetto et al 39 When looking at ratios of proteases to protease inhibitors, we did observe a negative correlation between MMP-2/TIMP-3 and wound reduction and MMP-2/TIMP-2 and wound reduction.…”

“…Biomarker correlation to percent wound reduction for: (A) TIMP-2, (B) MMP10, (C) EGF, (D) IL-1ra, (E) IL-7, and (F) IL-8. Red data points are from non-healing venous leg ulcers (VLUs) and blue data points are form healing VLUs including Eotaxin, GM-CSF, ICAM-1, IL-6, IL-16, MCP-1, MIP-1a, MMP-13, PDGF-BB, and TIMP-4 37. Similarly, Edsberg et al conducted a proteomic analysis on the wound fluid of pressure ulcers and identified 20 proteins which were also differentially expressed between chronic and healed wounds including GM-CSF, I-309, INF-γ IL-11, IL-12p40, IL-15, IL-1a, IL-1b, IL-8, TIMP-1, TIMP-2 TNF RI, TNF RII, ICAM-1, IL-16, MIP-1d, MMP-10, MMP-13, MMP-3, and Eotaxin-2.…”

Novel Cellulose Fibre-Based Flexible Plasmonic Membrane for Point-of-Care SERS Biomarker Detection in Chronic Wound Healing

“…Generally, a multi-omics approach to evaluating putative biomarkers associated with disease involves collecting biopsy samples from patients and transferring these to a laboratory for quantification using specialist techniques; for example, enzyme-linked immunosorbent assays (ELISAs) 5 or liquid chromatography–mass spectrometry (LC–MS). 8 This adds to lengthy analytical procedures as clinical samples have to be prepared in accordance with the assays employed.…”