had clotted, serum was separated from the sample by centrifuga- tion, 3000 rpm for 10 min. The plasma was immediately frozen in plastic tubes (Sarstedt, Reagiergefaße, micro tubes, 1.5 ml) at 220uC. Statistical analysis. Two tailed paired t-tests were used for comparison of different groups. All values are presented as mean 6 one standard deviation (SD), and a p-value,0.05 was considered significant. SigmaPlot 11 (Sy Stat Software/Inc.; Germany) was used for data analyses. Results Patients The study population consisted of fifty healthy volunteers, 36 males and 14 females with mean age 36.465.9 years (range 29–58) and mean weight of 77.5 kg. There were no complications to wick implantation or blood sampling. Wick fluid content and function of colloid osmometer The average wick length of 5 cm gave nearly 4 ml wick fluid after centrifugation. Two ml was used to measure colloid osmotic pressure, hence duplicate measurements were not possible. Each COP measurement took approximately 4 min. and was performed at laboratory temperatures of 20–22uC. COPC before testing patient IF showed a mean COPC of 27.361.5 mmHg (n = 77) with a variance of 2.2. Blood contamination of wicks From a total of 380 inserted wicks, 95 (22%) were discarded because of visible blood. The proportion of blood stained wicks was similar for wicks inserted without and with topical anaesthesia (23% vs. 21%) An additional 9.2% of the wicks had insufficient fluid content for analysis. Blood contamination occurred less frequently in subjects who only had three wicks inserted subcutaneously (13%). Sixty-six% of accepted wicks were classified as light pink, and the proportion of clear wicks was higher in the group receiving topical anaesthesia vs. no anaesthesia (83% vs. 71%). Measurement of plasma colloid osmotic pressure Mean COPp for all subjects was 27.661.8 mmHg (range 23.8– 31.8). Effect of equilibration time COPi in interstitial fluid isolated from dry and wet wicks at the different sampling times are shown in Fig. 1. For both dry and wet wicks COPi declined through the first 75 min. after implantation and subsequently increased slightly, but statistically not signifi- cantly until all were removed after 120 min. For dry wicks, mean COPi in the same tissue decreased from 25.964.4 mmHg after 30 min. of implantation to 21.161.5 mmHg after 75 min. (p = 0.043). At 120 min. the mean value was 23.765.0 mmHg (p.0.05 from nadir at 75 min.). A similar pattern was seen for wet wicks (21.063.2 mmHg at 30 min. vs. 18.763.0 mmHg at 75 min. (p.0.05) and 23.264.8 mmHg at 120 min (p.0.05) compared to 75 min.) Both dry and wet wicks reached an equilibrium level of 23 mmHg between 75 and 120 min. Effect of topical anaesthetics There were no significant differences between COPi from wet wicks implanted without or after application of a topical anaesthetic at any of the implantation times (Figure 2). After anaesthesia COPi slowly increased from 19.362.8 mmHg at Local Anaesthetics and Colloid Osmotic Pressure Figure 1. Colloid osmotic pressure and implantation time. Colloid osmotic pressure in interstitial fluid collected from wicks after various times of implantation. Significant difference (p,0.05) between dry (N) and wet (&) wicks at 30 min. are indicated with ( ) and between dry wicks from 30 to 75 min. with ( ). doi:10.1371/journal.pone.0031332.g001 30 min. to 21.964.4 mmHg at 120 min. while the corresponding figures without anaesthesia were 20.964.4 mmHg and 20.864.3 mmHg (figure 2). A significant lower VAS score was found comparing wick insertion with EMLA and no topical anaesthesia (4.2 vs. 5.8, p,0.0049) in a subgroup of 10 subjects. HPLC of isolated fluid/extravasation of plasma proteins The size distribution of proteins in fluids isolated from dry and saline soaked wicks and plasma were determined by HPLC. Representative elution patterns are shown in Fig. 3. Protein peaks in plasma corresponded to that of fluids from dry and wet wicks, and the elution pattern was similar in that the globulin peaks were higher than that of albumin. Except for a small haemoglobin peak, which was usually seen in samples from IF, no low molecular weight contaminants were detected. Figure 2. Topical anaesthesia and colloid osmotic pressure. Colloid osmotic pressure in interstitial fluid collected from wet wicks inserted without (N) or after topical anaesthesia with EMLA (m) plotted against duration of insertion. Mean COPi was 21.0 mmHg in the group without and 20.6 mmHg in the group with EMLA. No significant differences were found between the groups. doi:10.1371/journal.pone.0031332.g002 PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e31332