A simplified method for monitoring cytokines in wound fluid

Abstract

Cytokines in wound fluid are used as surrogates for wound healing in clinical research. The current methods used to collect and process wound fluid are noninvasive but not optimal. The aim of this prospective study was to evaluate a method (NovaSwab) by which wound fluid is collected by a surface swab and eluted in a physiological buffer for subsequent cytokine analysis. Wound fluid from 12 patients with leg ulcers was assessed by NovaSwab at the start (Day 0) and at the end of a 23-h collection period of wound fluid retained by foam oblates beneath an occlusive film dressing (Day 1). GM-CSF, IL-1α, IL-1β, IL-6, IL-8, PDGF-AA, TNF-α and VEGF levels were measured by multiplex and electrochemiluminescence assays. IL-1α (2.4×), IL-1β (2.0×) and IL-8 (1.8×) levels increased from Day 0 to Day 1 as detected by NovaSwab, indicating local production of these polypeptides in the wounds. On Day 1, the NovaSwab method yielded higher levels of IL-1α (4.0×), IL-1β (2.7×) and IL-6 (2.7×), and 35% lower levels of VEGF than those in wound fluid accumulated for 23 h in foam oblates (on average, 5 ml of wound fluid). In vitro experiments showed that the investigated cytokines in cell-free wound fluid were recovered in a quantitative manner by the NovaSwab method. We conclude that the method presented here is a promising research tool to study the kinetics of soluble cytokines over the course of wound healing. More studies are needed to determine the interobserver variation and reproducibility of the NovaSwab method.

Keywords: biomarkers; inflammation; leg ulcer; swab; wound fluid collection.

Conflict of interest statement

No company or organisation played any role in the initiation or design of the study, collection, analyses, or interpretation of the data, writing of the manuscript, or decision to publish the results. Reponex Pharmaceuticals sponsors Ewa Anna Burian's PhD studies through payments to the department. Ewa Anna Burian is investigating the effect of topical GM‐CSF in venous leg ulcers for Reponex Pharmaceuticals. Tonny Karlsmark is a medical advisor for Reponex Pharmaceuticals. Christian Enevold and Magnus S. Ågren declare no conflicts of interest.

© 2022 The Authors. Wound Repair and Regeneration published by Wiley Periodicals LLC on behalf of The Wound Healing Society.

Figures

FIGURE 1

The six‐step NovaSwab method for the measurement of cytokines in WF collected with a surface swab

FIGURE 2

Overview of WF samples collected from each patient. On Day 0, WF was obtained with surface swabs before (1st swab) and after saline irrigation (2nd swab). Foam oblates were then applied to the wound bed and the wound was covered with an occlusive film dressing according to Zillmer et al. On Day 1, WF was squeezed out from the foam oblates and centrifuged to yield a cell‐free supernatant (controls in the in vitro tests). Finally, WF was collected with a 3rd swab.

FIGURE 3

Cytokine levels normalised to the total protein measured in WF accumulating from Day 0 to Day 1 for 23 h in foam oblates (Day 0 → 1) and by the NovaSwab method (Day 1). (A) Individual and mean cytokine values from the 12 patients. *p < .05; **p < .01. Triangles, WF collected by foam oblates; open circles, WF samples obtained by eSwab (first 6 patients); filled circles, ∑‐Transwab (last 6 patients). (B) Bland–Altman plots of agreement between cytokine levels in WF determined with the NovaSwab method on Day 1 and WF accumulating in foam oblates from Day 0 to Day 1 (control). The mean bias is indicated by the horizontal grey lines, and the 95% limits of agreement are indicated by the dotted horizontal lines.