Fresh porcine blood was sourced from a local slaughterhouse (Münchner Schlachthof Betriebs GmbH, Munich, Germany) and processed following a standardized protocol. The blood was mixed with a modified Ringer's solution to attain a hematocrit level of 36%, maintaining standard electrolyte concentrations and typical blood gas values. To prevent coagulation, we administered 30,000 IU of heparin per liter of blood (Ratiopharm, Ulm, Germany). The blood was maintained at a constant temperature of 37 °C and gently agitated at a rate of 130 rpm to ensure its suitability for our intensive care research.

Different concentrations of IL-6 were spiked in blood. Briefly, recombinant human IL-6 (PeproTech, Cranbury, NJ, USA) was prepared according to the manufacturer's instructions for handling and reconstitution. The IL-6 was initially reconstituted in distilled water by gently shaking the vial after centrifugation. The IL-6 reconstituted solution was stored at 2 °C for short term use (up to 1 week) or at − 20 °C to ensure long term stability. During experiments, 0.5, 2.5, 10 and 50 µg of IL-6 were spiked into 5 L blood to achieve IL-6 concentrations of 100, 500, 2000 and 10,000 pg/mL, respectively.

The ADVOS multi (ADVITOS GmbH, Munich, Germany) is a hemodialysis system intended for the removal of water-soluble and protein-bound substances, for the correction of blood composition in case of electrolyte imbalance, including metabolic or hypercapnic acidosis, and for the removal of fluid, if needed. It consists of three interconnected circuits (Fig. 1). Briefly, bloods flows (100–400 mL/min) through the extracorporeal circuit, which bears two ELISIO 19H dialyzers (Nipro D.Med Germany GmbH, Hamburg, Germany). Here, human albumin enriched (200 mL, 20%) dialysate flowing from the dialysate circuit (800 mL/min) receives toxins from blood. The toxin-loaded dialysate fluid enters and recirculates then through the ADVOS multi regeneration circuit. Here, by applying pH and temperature changes, dialysate albumin adapts its structure to release the protein-bound toxins. These are then filtered by convection via two ELISIO 13H filters (Nipro D. Med Germany GmbH, Hamburg, Germany) into the waste together with the water-soluble toxins. The removed volume is replenished with fresh dialysate (160–320 mL/min), which is obtained by continuously mixing osmosis water, an alkaline (i.e., mainly NaOH), and an acidic concentrate (i.e., mainly HCl) online. Finally, the acidic to alkaline concentrate ratio defines the individualized dialysate pH (7.2–9.5).

Schematic representation of ADVOS multi and the experimental setting. A blood pool of 5 L was spiked with different concentrations of IL-6 and was subsequently treated with ADVOS multi. The ADVOS hemodialysis system consists of an extracorporeal circuit, a dialysate circuit and an albumin regeneration circuit (i.e., ADVOS multi circuit)

Additionally, in order to maintain adequate and physiological blood gas values, CO2 was continuously infused into the blood pool via an additional ELISIO 19H dialyzer (Nipro D.Med Germany GmbH, Hamburg, Germany) connected to a CO2 gas supply (Linde AG, Munich, Germany), as previously described [13].

In a preliminary analysis to rule out a spontaneous change of IL-6 levels, blood was spiked with a known concentration of IL-6. Blood was continuously stirred for 4 h without further treatment. Duplicate samples were taken and analyzed at different timepoints. Blood without IL-6 addition was used as negative control.

Once stated that no decomposition of IL-6 occurred in our blood model, blood was subjected to 4-h treatments with ADVOS multi. Table 1 shows the different settings that were employed with the aim to determine the effect of varying IL-6 concentrations, and blood and concentrate flows on the reduction rate and clearance of IL-6. Finally, to state if IL-6 could be continuously removed beyond 4 h, treatments lasting 12 h were conducted with a starting concentration of 10,000 pg/mL IL-6.

Blood samples were taken in duplicate at minutes 0, 15, 30, 60, 120, 180, and 240 (4 h), and additionally at minutes 480, 720 (12 h) for the longer experiments. Each experiment was performed 3 times.

Blood and dialysate samples (5 mL) were obtained at the specified time points. Blood was centrifuged at 4000 rpm for 15 min at 4 °C and 1 mL of the blood plasma was stored at − 25 °C until analysis. Dialysate did not undergo centrifugation. The Elecsys IL-6 sandwich chemiluminescent immunoassay (Roche Diagnostics GmbH, Mannheim, Germany) was used for sample analysis. Briefly, a sample of 18 µL is initially mixed with IL-6 specific antibodies and then incubated with ruthenium labelled IL-6 specific antibodies to form a sandwich complex. The complexes are then magnetically captured, inducing a chemiluminescent emission proportional to IL-6 concentration.

Reduction rate was calculated as stated in Eq. 1, where Cf refers to the final IL-6 concentration (e.g., at 240 or 720 min for 4 and 12 h experiments, respectively), and C0 is the started spiked IL-6 concentration.

$$\text \left(\%\right)=1-\frac_}_} \times 100$$

(1)

Equation 2 was employed for the calculation of the elimination constant (Ke), where tf and t0 reflect the final (i.e., 4 or 12 h) and the starting (i.e., 0 min) timepoints, respectively. Ke was calculated assuming a first order kinetics.

$$_(^)=\frac(\frac_}_})}_-_)}=\frac_-\text_}_-_)}$$

(2)

For clearance (CL) calculation, a volume of distribution (Vd) of 5 L (i.e., blood volume) was set within Eq. 3.

$$\text (l/h)=\text\times _$$

(3)

Data were documented and analyzed using Microsoft Excel and IBM SPSS 28.0 for Windows®, respectively. Reduction rate, and clearance are presented as mean and standard deviation (SD). An analysis of variance (ANOVA) followed by Bonferroni tests was conducted to determine differences between varying treatment settings. A p value lower than 0.05 was considered to indicate statistical significance.