Donor skin
Following donor consent, full thickness human skin was obtained from three women aged 51, 52, and 53 years. All skin was excess abdominal tissue following deep inferior epigastric perforators (DIEP) flap surgery for breast reconstruction and was frozen at −20 °C on the day of excision and used within four weeks. Ethics committee approval was granted by NRES Committee West Midlands, UK (REC: 14/WS/1012). All skin donors consented for their tissue to be used in research.
Skin penetration of CHG using an ex -vivo skin model
Studies were performed as in previous ex vivo skin penetration investigations undertaken by our group [3, 7]. In brief, the receptor compartments of Franz diffusion cells were filled with 15 mL of sterile PBS and maintained at 37 °C. Eighteen pieces of full thickness skin (six pieces from each of three donors) measuring 3 cm × 3 cm were thawed before mounting onto the cells with the stratum corneum facing upwards. Any entrapped air between the skin and the receptor fluid was removed. The skin surface was blotted dry and left to equilibrate for 30 min. Two of the skin samples from each donor had 100 μL of a solution containing 2% (w/v) CHG (Sigma Aldrich, Poole, UK) and 70% (v/v) IPA (Fisher Scientific, Loughborough, UK) applied to the surface and two skin samples from each donor had 100 μL of a solution containing 2% (w/v) CHG, 70% (v/v) IPA and 2% (v/v) 1,8-cineole (Sigma Aldrich, Poole, UK) applied. The solutions were spread evenly across the skin surface (3.14 cm2) and left at room temperature for 24 h. The two remaining skin samples from each donor were mounted onto the Franz cells without the application of antiseptic. These were used to quantify background CHG levels present in the skin as all donor patients received pre-operative skin preparation with 2% (w/v) CHG in 70% (v/v) IPA [ChloraPrep® CareFusion UK Ltd., Basingstoke, UK] prior to skin excision.
The skin samples were frozen with a cryospray (Leica, Milton Keynes, UK) and three punch biopsies (7 mm in diameter, totalling an area of 1.15 cm2) removed from each sample. This equated to approximately 732.5 μg of CHG applied to each punch biopsy area. In comparison, when ChloraPrep® one-step applicators are used in clinical situations, according to the manufacturers’ instructions, a minimum of 22.98 μL 2% (w/v) antiseptic solution (equal to 459.6 μg CHG) per cm2 of skin is delivered. The solution used in our study therefore provided a comparable level of CHG to that by ChloraPrep® in the clinical scenario. Skin biopsies were subsequently sectioned horizontally with a cryotome (Leica, Milton Keynes, UK) into 100 μm sections (to a depth of 2000 μm to encompass some depth of follicular sectioning). Corresponding sections from the three biopsies were pooled in separate sterile polypropylene centrifuge tubes (Fisher Scientific, Loughborough, UK) and the weight of each sample determined.
Quantification of CHG
Quantification of CHG was determined using a validated high performance liquid chromatography (HPLC) method as described previously [3, 7]. Extraction of CHG from skin sections was carried out by the addition of 1 mL of HPLC isocratic mobile phase [75% (v/v) HPLC grade methanol (Fisher Scientific), 25% distilled water, 0.1% (v/v) HPLC grade diethylamine (Sigma Aldrich), and 0.005 M sodium heptane sulphonate (Sigma Aldrich) adjusted to pH 4 with HPLC grade glacial acetic acid (Fisher Scientific)] to each centrifuge tube containing corresponding sections from three biopsies which were incubated at 37 °C for one hour. All samples were then vortex-mixed for 30 s, centrifuged for 10 min at 6000 g, filtered through a 0.45 μm filter, and the CHG quantified with an Agilent 1200 series HPLC system (Agilent Technologies, Stockport, UK). Samples were run at room temperature at 1.2 mL/min through a reverse-phase chromatography column [CPS-2 Hypersil 5-μm column; 150 mm × 4.6 mm (Fisher Scientific)] with UV detection at 254 nm. CHG extraction was validated prior to the study and HPLC with every run [3, 7].
Statistical analysis
To improve the validity of assumptions of normal distributions, the 2% (w/v) CHG and 70% (v/v) IPA, 2% (w/v) CHG and 70% (v/v) IPA with 2% (v/v) 1,8-cineole and background values at each depth were log transformed. These values were used in the statistical analysis. The data were analysed with repeated measures analysis of variance with two within subjects factors, a) background, with and without 1,8-cineole and b) depth below the skin surface. The Greenhouse-Geisser test was used to assess the significance of F values. Repeated contrasts were used to compare concentrations at successive depths. SPSS version 22 (IBM, Armonk, NY) was used for data analysis.
