**2.1 Methods for leukocyte functional studies**

The method used was the skin chamber technique, which is well documented and has been used by a number of investigators to study transmigration and recruitment of leukocytes at the inflammatory site (Scheja & Forsgren 1985; Follin 1999; Thylen et al. 2000; Jacobson et al. 2002; Theilgaard-Monch et al. 2004; Dadfar et al. 2007; Paulsson et al. 2007). With the skin chamber technique, we measured leukocyte functions at time 0 (before the high-flux hemodialysis/hemodiafiltration session) and after 10 hours (within which time the highflux hemodialysis/hemodiafiltration treatment was performed). The terms intermediate and intense inflammation were used to designate the blister stimulated with buffer and with autologous serum, respectively.

Leukocytes were measured with flow cytometry or FACS (fluorescence-activated cell sorting) a method in which cells are scanned by a laser and recognized as different cell populations through their light-scattering properties. Different leukocyte populations (lymphocytes, monocytes and neutrophils) can thus be counted and expressed as a percentage of the total leukocyte population. Mean fluorescence intensity (MFI) values for the different analyses of cell functions (CD11b expression, hydrogen peroxide formation and apoptosis) can also be measured and quantified.

The CD11b expression on leukocytes, both unstimulated and after stimulation with fMLP, was studied through immunostaining. Analysis of leukocyte hydrogen peroxide formation, after stimulation with fMLP or PMA, was performed using the 2', 7'-dichlorofluorescein diacetate (DCFH-DA) method. We also stained leukocytes with Annexin V and propidium iodide (PI) to identify cells that were in an early or late apoptotic state.

 Median 25%-75%

Patients Non-Outlier Range

p < 0.001

Healthy PMA

Patients PMA

Healthy

0

significant difference is present.

20

40

60

80

100

120

140

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180

Patients fMLP

Fig. 3. Respiratory burst in neutrophils at the site of an intermediate interstitial inflammation expressed as mean fluorescence intensity (MFI). P is indicated where a

Healthy fMLP

Chemokines in skin blister fluids and serum from the peripheral circulation were analyzed with commercially available immunoassays (Quantikine®, R&D Systems Inc. Minneapolis, MN, USA). All immunoassays were used in accordance with the manufacturer's instructions. For further details, please review publications (Olsson et al. 2007 & Olsson et al. 2009).
