629 Validation of a wound fluid collection method for subsequent identification of prognostic biomarkers for healing

Abstract

Several matrix metalloproteinases (MMPs) are hypothesized to play key roles in the wound healing process due, to their ability to break down various extracellular matrix components. Expression levels of MMPs, or their inhibitors, may be useful for predicting the risk of wound healing or non-healing. However, methods used to collect human wound fluid are diverse with little evidence of rigorous method validation in the literature. Therefore, we aimed to develop a robust method for the collection of fluid from wound dressings. We have validated this method using both human serum and wound fluid samples. Serum was acquired commercially whilst wound dressings were collected from ten patients undergoing treatment of leg or foot ulcers. Total protein concentrations and relative protein abundances were measured for extracted and control samples. The developed method gave normalised total protein recoveries (arbitrary units, a.u.) of 0.64, 0.85, 0.60 and 0.56 a.u. when extracting serum from alginate (Kaltostat), foam (Mepilex), surgical (Softpore) and film (Tegaderm) dressings respectively in comparison to serum controls (1.00 a.u.). Fluid extracted from patient dressings had a mean total protein concentration of 21.5 mg/ml and gave consistent identification and quantification of several MMPs and their inhibitors via Western blot. The developed method therefore facilitated consistent recovery of serum with minimal protein loss, specifically when using foam dressings. However, these data show the variability in total protein recovery between dressing materials. This method’s suitability was further demonstrated with the successful extraction of wound fluid from patient dressings and quantification of proteins involved in wound healing. This method is therefore suitable for further wound fluid collection procedures and development of prognostic biomarker studies.