understanding and treatment of various disease processes [8] [9] [10]. Some methodological work has been performed in volunteers, with the assumption that similar correlations occur in healthy humans [11] [12]. Implantation of wicks in adults is usually done after intradermal injections of a local anaesthetic, and we know from pilot studies that without local anaesthetic, the wick insertion causes some pain and discomfort. To our knowledge, there are no data regarding COPi in children, mainly because this patient group has not been available for such non-therapeutic research. Minimizing both unpleasant and distressing procedures and trauma from physical and emotional pain is obligate when a paediatric population is involved in clinical research [13]. In order to apply the wick technique for sampling of IF in children it is therefore important to examine how the application of topical anaesthetics on the skin affects the measured values of COP in the IF. The aim of this study was therefore to evaluate whether topical application of anaesthetics affects measured values of COPi. In addition we wanted to examine whether measured COPi is different when IF is obtained from dry or saline soaked wicks and to validate optimal wick implantation time in healthy humans. Materials and Methods Ethics Statement The protocol for this study was approved by the local ethics committee (Regional Committee for Medical and Health Re- search Ethics, Western-Norway) at the Department of Paediatrics, Haukeland University Hospital, where all human experimentation was conducted. Participants were continuously recruited from the clinical staff after simple enquiry. Written informed consent was obtained from all participants involved in the study. The wick method Sampling of IF for determination of COPi and protein distribution were achieved by means of multifilamentous nylon wicks with a diameter of about 0.8 mm (Polyamid no. 8, Norsk Fletteri AS, Bergen, Norway) sewn into subcutaneous tissue. All wicks were sterilized by gamma irradiation (Institute for Energy Technology, Kjeller, Norway). Double threaded wicks were placed on straight sterile suture needles (Acufirm, 210/3, Germany) and tied with a knot at the end. The skin was disinfected with a solution of 0.5% chlorhexidine before inserting the wick in lengths of approximately 5 cm. Except for the knot, ends protruding from the skin were cut of, leaving the knot with 0.5 cm of wick on the outside. Adhesive plastic film (Tegaderm, 3 M Canada Inc.) was placed over both ends of the wick in order to reduce evaporation of fluid. All wicks were placed in parallel, and with a distance of at least 1 cm to each other. At the end of the implantation period, the central part of each wick was rapidly transferred to separate mineral oil filled centrifuge tubes with funnel. Heavily blood stained wicks (judged visually) were discarded and blood stained parts of wicks were cut off if possible. Wicks were centrifuged for 10 minutes at 14.000 rpm and wick fluid could then be collected at the bottom of the tube because the plastic funnel held back the wick during centrifugation. All samples were frozen in plastic tubes (Sarstedt, Reagiergefaße, micro tubes, 1.5 ml) at 220uC until analysis. All procedures were performed under sterile conditions. Before analysis, room tempered samples were transferred by pipette to a glass capillary tube. The capillary tube was sealed in one end with plasticine (Haematocrit sealing compound, No.74950; BRAND, Germany) and centrifuged in a haematocrit centrifuge (SABA, Haematocrit 20, Hettich) for 3 min. in order to separate mineral oil from wick fluid. Local Anaesthetics and Colloid Osmotic Pressure Colloid osmotic pressure measurements COP was measured directly with a semi permeable membrane transducer colloid osmometer designed for small fluid samples [14] [15]. The reference camber was filled with isotonic saline (NaCl 9 mg/ml) and a membrane impermeable for molecules greater than a molecular weight of 30-kDalton (PM-30 Amicron, Lexington, MA, USA) was used. Registration of negative hydrostatic pressure, when samples were put on top of the membrane, was measured by a pressure transducer, amplified and recorded (Easy Graph P930, Gould Inc., USA). Before analysing samples the colloid osmometer was calibrated hydrostatically with a saline filled column representing a pressure of 20 mmHg. In order to verify membrane response and to test the accuracy of the whole system, a solution of serum with a known colloid osmotic pressure (COPc) was used as a standard reference. After flushing the sample chamber 4 times, 2 ml saline was left for recording zero pressure. Both saline, serum and wick fluid were sucked up by a soft absorbing tissue paper. High performance liquid chromatography (HPLC) In order to determine the distribution of macromolecules in the IF and plasma, a high-resolution size exclusion chromatography using a 4.6 mm (ID)630 cm TosoHaas Super Sw3000 (Tosoh Biosciences, Stuttgart, Germany) with an optimal separation range for globular proteins of 10–500 kDa was used. One ml of isolated plasma or wick fluid was diluted in 49 ml HPLC buffer to a total volume of 50 ml, and 5 ml of this solution was injected onto the column using an A-905 auto sampler connected to an Ettan 900 LC System (GE Healthcare, formerly Amersham Biosciences). The column was eluted at a constant flow of 0.35 ml/min (P-905 pump) with a 0.1 mM phosphate buffer (pH 6.7) containing 0.1 mM Na2SO4. Proteins were measured by UV detection at 210 nm using an UV-900 monitor and a 0.7 ml flow cell (path length of 3 mm) connected directly to the column outlet. The HPLC-system was operated by a unicorn software module (ver. 4.12). Experimental design The study was designed as a non-blinded, sequential descriptive study. The study population was enrolled between March 2008 and September 2010. Effect of equilibration time. In order to compare equilibrium time for dry and saline soaked wicks, four dry nylon wicks were inserted subcutaneously into one upper arm and four saline soaked wicks into the contra lateral upper arm in 20 subjects. Thereby, each subject served as his/her own control. Implantation time was 30, 60, 90 and 120 min. on both sides. Ten additional subjects had 3 wicks implanted for 60, 75 and 90 min. respectively. Effect of topical anaesthetics. In order to study a possible effect of topical anaesthetics on the COPi measurements saline soaked wicks were used in 20 subjects. Saline soaked wicks are commonly used in human studies [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] and from our own pilot studies, they cause less pain than dry wicks during insertion. Four wicks were inserted subcutaneously in the same position on each upper arm. On the test arm 2.5 grams EMLA cream was applied and covered with an occlusive dressing for 60 minutes prior to insertion of the wicks (1 gram equals a narrow strip that is 38 mm65 mm wide containing lidocaine 2.5%/prilocaine 2.5%). Placebo cream was not applied on the control arm before insertion of the wick. During insertion, ten research subjects graded pain on a visual analogue scale (VAS). A venous blood sample of 5 ml was obtained from all participants after the wicks had been inserted. After the blood PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e31332