Wound Fluid Collection Protocol for CVLU

Wound fluid is collected from unhealed ulcers at each study time point using a standard wound fluid collection protocol [25 (link), 29 (link)]. The fluid is collected by the PI experienced in the protocol or CRC nurses trained by the PI. Briefly, after CVLUs are washed with sterile water, a transparent occlusive film (Opsite, Smith & Nephew, UK) is applied over the wound and the leg is placed in a dependent position for approximately 1–1½ hours. While slowly removing the occlusive film and rinsing the wound with 1 ml of sterile saline, the fluid is collected using a 26G × 0.5″ angiocatheter attached to a 3-ml syringe (Terumo Medical, Somerset, NJ, USA). The fluid is transferred into plain collection tubes and analyzed immediately to determine PMN activation or frozen and stored at − 80^° C until further analysis.