A target-orientated algorithm for regional citrate-calcium anticoagulation in extracorporeal therapies.

BACKGROUND: Citrate anticoagulation offers several advantages in comparison to conventional anticoagulation. Most algorithms for regional citrate-calcium anticoagulation are based on citrate and calcium chloride infusion coupled in a fixed proportion to the blood flow without considering the hematocrit (Hct)/plasma flow or the filter clearance of citrate and calcium. METHODS: The aim of this study was to develop an algorithm for optimized citrate anticoagulation in extracorporeal therapies such as dialysis. A mathematical model was developed to calculate the volume of citrate infusion required to achieve a desired ionized calcium (iCa) target level in the extracorporeal circuit and to restore the total calcium level to a physiological value. RESULTS: The model was validated by correlation analyses for different blood Hct values and shows an excellent fit to the laboratory measurements. CONCLUSION: The results for both iCa target concentrations, namely those after citrate and calcium infusion, proved that the software algorithm adapts well to variable treatment parameters.

Anticoagulation in combined membrane/adsorption systems.

For extracorporeal blood purification treatments, an effective anticoagulation is needed to avoid contact activation via the intrinsic pathway of the blood-clotting system. While heparin is the standard anticoagulant in dialysis, it shows some disadvantages which have to be considered when it is used in membrane/adsorption-based blood purification systems. An alternative option for anticoagulation in these systems is citrate, which is effective as an anticoagulant by reducing the ionized calcium concentration in the extracorporeal circuit. However, to avoid citrate accumulation in the patient during treatment, the amount of citrate infusion and the citrate removal by the patient's metabolism as well as by dialysis have to be taken into consideration. The aim of this study was to elucidate the characteristics of heparin removal in membrane/adsorption-based blood purification systems, to find the correct way to pre-coat adsorbents in order to avoid excessive adsorption of heparin by anionic exchanging resins, and also to find an appropriate dosage of heparin for treatments with these systems to ensure patient safety. A further aim was to find the correct ionized calcium concentration to suppress complement activation, and to compare different dialysis filters regarding their citrate clearance in order to be able to recommend the correct dialysis setup to achieve appropriate citrate clearance. We were able to show that the adsorptive removal of heparin can be significantly reduced by pre-coating the adsorbents with heparin without a perceptible impact on the adsorption kinetics of bilirubin. Furthermore, we recommend the use of unfractionated heparin due to its lower sieving coefficient and therefore lower removal compared to fractionated heparins. Reducing the extracorporeal Ca(2+) concentration to 0.2 mmol/L by infusion of citrate solution to the extracorporeal circuit results in an effective suppression of the complement activation. To avoid citrate accumulation, we recommend the use of high flux filters when citrate anticoagulation is applied.

New methods for hemoglobin detection in a microparticle-plasma suspension.

In the extracorporeal adsorption system, MDS (Microspheres based Detoxification System), micro-adsorbent particles measuring 1-25 micrometers circulate in a filtrate circuit for highly specific blood purification/adsorption. The MDS circuit containing the adsorbent microparticles is linked to the patient's blood line by a hollow fiber plasma filter. When the transmembrane pressure or the shear forces due to the red blood cells in the hollow fiber filter are too high, they can be damaged and hemoglobin will be released. In order to detect free hemoglobin (fHb) by optical means, we have designed a new flow-dynamic filter system, placed in the microadsorbent circuit for continuous separation of microparticles from the filtrate. In the flow dynamic filter, we use a high velocity liquid vortex to remove sedimentation and particle plugs on the filter membrane. In our investigations, 3 and 8 micron cellulose nitrate filter membranes for particle separation are used. The obtained particle free bypass filtrate flow rates are typically 0.5 and 0.8 ml/min respectively. The typical sensitivity for fHb detection by the applied noninvasive optical method is 0.15 g/dL. Medical safety regulations require a fail-safe mechanism for fHb detection which monitors the bypass filtrate flow in the flowdynamic filter and shuts down the system in case of membrane occlusion. The bypass filtrate flow is monitored by periodically occluding and releasing the bypass line by means of a clamp. The resulting back pressure profile gives information about the actual filtration rate. This safety principle was proven by statistical analysis and shows its clear functionality.

Comparison of specific adsorbents for tumor necrosis factor-alpha and therapeutic anti-tumor necrosis factor-alpha antibodies: an in vitro sepsis model.

BACKGROUND: Tumor necrosis factor alpha (TNF-alpha), as a key mediator, represents a major point of attack in sepsis. Since it has been shown that systemic anti-TNF-alpha antibodies do not improve the situation of septic patients, the use of specific adsorption technology in the treatment of sepsis could have beneficial effects. METHODS: Magnetic beads coated with polyclonal or with monoclonal anti-TNF-alpha antibodies were investigated in vitro in order to analyze their ability to prevent TNF-alpha induced adhesion of peripheral blood mononuclear cells (PBMCs) at human umbilical vein endothelial cells (HUVECs). Additionally, therapeutical monoclonal anti-TNF-alpha antibodies were proofed for inhibitory effects of TNF-alpha mediated activation of HUVECs. RESULTS: We have shown, in vitro, that beads coated with polyclonal or monoclonal anti-TNF-alpha antibodies were able to significantly reduce monocyte adhesion. It was possible to decrease monocyte adhesion from nearly 9% to 3% within 2 hours and from 18% to 2% within 6 hours of TNF-alpha treatment by the simultaneous use of beads coated with polyclonal anti-TNF-alpha antibodies. Beads coated with monoclonal anti-TNF-alpha antibodies could even prevent monocyte adhesion within the first 2 hours, and reduced monocyte adhesion to 2% during 6 hours of incubation with TNF-alpha. On the other hand, application of therapeutic anti-TNF-alpha antibodies showed no significant difference compared to the measured monocyte adhesion values of activated endothelial cells. CONCLUSION: Adsorption techniques using specific adsorbents, possibly used in MDS (Microspheres-Based Detoxification System), are efficient in specific reduction of TNF-alpha and pathophysiological consequences, since monocyte adhesion at activated HUVECs was shown to be reduced.

Detection of fluorescently labeled microparticles in blood.

BACKGROUND: A microsphere-based detoxification system is an adsorption system, whereby microadsorbent particles having diameters of 1-20 microm circulate in an extracorporeal filtrate circle. A thin-wall hollow-fiber membrane filter separates the microparticle-plasma suspension from the bloodstream. For patient safety, it is necessary to have a means to detect membrane ruptures that could lead to a release of microparticles into the patient's bloodstream. METHODS: An optical detection system was developed to monitor the venous bloodstream for the presence of microparticles from the filtrate circuit. For detection purposes, cellulose microspheres, both ferromagnetic and fluorescence labeled, were included with the microsphere adsorbant particles. In the case of a membrane rupture, the labeled particles would also be released into the bloodstream. By illuminating a small volume of blood with an excitation wavelength (590 nm) of the fluorescence marker, the particles can be detected by their emission light at 620 nm. The detector sensitivity is increased by collecting the ferromagnetic and fluorescently labeled microparticles using a magnetic trap. The efficiency of magnetic trap arrangements was tested by adjusting the magnet placements. RESULTS: In vitro experiments were performed by pumping whole blood and labeled microparticles through the fluorescence detector. The efficiency of a magnetic trap arrangement was determined. With an optimal trap setup, 5-10 microl of labeled microparticles can be clearly detected in streaming whole blood. CONCLUSION: An easy to handle microparticle detector was developed, ready for use in particle based blood detoxification systems. The microparticle detection system fulfills the medical and technical requirements to bring the MDS into clinical tests.

Patient safety technology for microadsorbent systems in extracorporeal blood purification.

Alternative technologies for extracorporeal blood purification systems based on microadsorbents in suspension are discussed. Principally, microadsorbents offer higher efficiency and flexibility when compared to conventional column-based adsorption systems. Systems already clinically employed (e.g., BioLogic DT) or close to clinical application (e.g., the microspheres-based detoxification system, MDS) are described. The MDS technology, in particular, is characterized by efficiency and a high degree of flexibility with respect to both the use of different adsorbents as well as the combination with hemodialysis/hemofiltration therapy. It was designed for continuous use in intensive-care units, but enables also the removal of low-density lipoprotein, fibrinogen, autoimmune antibodies, immune complexes, and other pathophysiologically relevant substances. Alternative anticoagulation regimes and safety systems on fluorescence sensor technology have recently been developed for the MDS and are presented in this paper.

Cellular interleukin-1 receptor antagonist production in patients receiving on-line haemodiafiltration therapy.

BACKGROUND: Repetitive exposure to cytokine-inducing substances (pyrogens) results in chronic inflammation, which may significantly contribute to some of the long-term complications in dialysis patients. On-line dialysis modalities, such as on-line haemodiafiltration (HDF), raise particular concerns because of the administration of infusate prepared from potentially contaminated dialysis fluid. Hence, great retention capability for pyrogens is of critical importance for the safe performance of on-line systems. METHODS: The microbiological safety of a novel on-line system, ONLINEplus(TM), was assessed in clinical practice in five centres for 3 months. Infusate and dialysis fluid were regularly monitored for microbial counts, endotoxins, and cytokine-inducing activity. Levels of interleukin-1 receptor antagonist (IL-1Ra) were determined in supernatants of whole blood incubated either under pyrogen-free conditions (spontaneous cytokine production) or following low-dose endotoxin exposure (LPS-stimulated cytokine production). RESULTS: We failed to detect microorganisms or endotoxin contamination of infusate during the entire study period. Moreover, neither infusate nor dialysis fluid demonstrated cytokine-inducing activity. Intradialytic IL-1Ra induction was not detected, as there was no difference between pre- and post-session values for both spontaneous and LPS-stimulated IL-1Ra production (115+/-26 vs 119+/-27 and 2445+/-353 vs 2724+/-362 pg/10(6) white blood cells (WBC), respectively). Neither the number of immunocompetent cells nor their capacity to produce IL-1Ra declined during this period, indicating that cells were not significantly stimulated during treatment. Spontaneous and LPS-induced exvivo IL-1Ra generation remained unchanged after 3 months of on-line HDF therapy as compared with the start of the study (71+/-30 pre- vs 48+/-14 post-study, and 2559+/-811 vs 2384+/-744 pg/10(6) WBC, respectively). CONCLUSIONS: The present on-line system performed safely from a microbiological view-point as both the dialysis fluid and infusate were consistently free of microorganisms, endotoxins, and cytokine-inducing substances. As a result, on-line HDF therapy had no effect upon the chronic inflammatory responses in end-stage renal disease patients.

Development of affinity microparticles for extracorporeal blood purification based on crystalline bacterial cell surface proteins.

In this article, the development of specific adsorbents for extracorporeal blood purification are described. Affinity microparticles were prepared by linking Protein A to crystalline cell surface layers (S-layers) from Thermoanaerobacter thermohydrosulfuricus 1111-69. S-layers were used in the form of cell wall fragments obtained by breaking whole cells by ultrasonification, resulting in cup-shaped structures (average size 0.5 x 1 microm) completely covered with S-layer protein. Protein A was covalently bound to carboxylic acid groups of the S-layer protein after activation with 1-ethyl-3,3'(dimethylamino)propylcarbodiimide. In batch adsorption experiments with fresh frozen human plasma, the resulting S-layer based affinity microparticles showed a high adsorption capacity for IgG (40 mg IgG were bound per g wet pellet of S-layer based affinity microparticles). Fractions eluted from the microparticles were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They contained only IgG demonstrating that adsorption was specific. In biocompatibility tests, preparations of the S-layer microparticles showed no low-density lipoprotein-reactivity, no cytotoxicity, and no cytokine inducing activity.

Cytokine induction in patients undergoing regular online hemodiafiltration treatment.

End-stage renal disease (ESRD) patients are known to suffer from chronic inflammation as the result of an ongoing subacute cytokine induction, which may contribute considerably to dialysis-related, long-term morbidity and mortality. Preparation of infusate from cytokine-inducing dialysis fluid and its administration in large quantities as well as the use of high-flux membranes bear the risk of aggravating the chronic inflammatory response among online hemodiafiltration (online HDF) patients. In order to assess the inflammatory risk associated with online HDF, we compared the cytokine induction profile of ESRD patients receiving either online HDF or low-flux hemodialysis (low-flux HD). Specifically, we measured spontaneous and lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNFalpha) and interleukin-1 receptor antagonist (IL-1Ra) release during ex vivo incubation of whole blood. Ultrapure dialysis fluid and polysulfone membranes were used for both treatment modalities. LPS-stimulated release of TNFalpha and IL-1Ra was elevated for both online HDF and low-flux HD patients compared to healthy individuals (TNFalpha: 2,336 +/- 346 and 2,192 +/- 398 versus 1,218 +/- 224 pg/106 white blood cells [WBC]; IL-1Ra: 2,410 +/- 284 and 2,326 +/- 186 versus 1,678 +/- 219 pg/106 WBC). Likewise, spontaneous production of TNFalpha, but not IL-1Ra, was higher in online HDF and low-flux HD patients than in normal controls (37 +/- 32 and 22 +/- 19 versus 0.8 +/- 0.3 pg TNFalpha/106 WBC). There was no difference in spontaneous and LPS-stimulated cytokine release between both dialysis groups. In addition, intradialytic cytokine induction was not significant for either treatment modality as spontaneous and LPS-stimulated cytokine release were not increased postdialysis. These findings indicate that online HDF does not contribute to chronic leukocyte activation and, consequently, does not place ESRD patients at greater risk with respect to inflammatory morbidity and mortality.

A novel configuration of bioartificial liver support system based on circulating microcarrier culture.

The purpose of this investigation is to initiate a new bioartificial liver support system that utilizes circulating microcarrier cultures in the extracapillary space of a hollow fiber cartridge. The material exchange occurs on the membranes of the hollow fiber. Toxins are metabolized by the circulating cells on the microcarriers driven by a centrifugal pump. We inoculated 2-3 x 10E8 Hep G2 cells on 2.5 grams of Cytodex 3 microcarriers, and allowed them flowing in the extracapillary space of a modified plasma filter. 10% FCS Medium was pumped through the capillaries at different rates. Cells keep morphological integrity and functionality during the circulation. These preliminary results suggest that this configuration of a bioartificial liver support system offers a future investigation.

Blood purification by a membrane technique--a new method for the effective removal of low-density lipoprotein cholesterol.

A membrane plasma fractionation (MPS) technique is applied in order to obtain selective removal of pathological plasma components from the extracorporeal circuit. An effective plasma fractionation procedure should be characterized by the highest possible removal of the pathological plasma components and, equally as important, the lowest unwanted protein losses caused mainly by adsorption to the membrane structure. In order to obtain a higher efficiency of the MPS procedure (high selectivity between removal of pathological plasma components and unwanted losses mainly represented by albumin) several methods such as thermofiltration, application of pulsate flow at the end of secondary filter, etc. have been developed. Clinical verification of these methods led to some improvement in MPS procedure but these results did not seem to be optimal. The main objective of this paper is to present a new two-stage membrane system utilizing a high flow recirculation circuit developed particularly, but not only, for effective removal of low-density lipoprotein cholesterol. The designed and developed system has been tested in vitro using several different plasma fractionation membranes. The results obtained indicated the importance of the membrane structure and membrane material on the efficiency of the tested plasma fractionation procedure. It was also found that it is possible to obtain negligible protein losses for some selected membrane structures applied in the assessed system. Based on preliminary results, it seems that the new two-stage membrane system proposed could be characterized by a very low range of unwanted protein losses leading to high effectiveness of the plasma fractionation procedure.

Novel online infusate-assisted dialysis system performs microbiologically safely.

Administration of adequate amounts of commercial infusion fluids renders modern convective dialysis modalities, such as hemodiafiltration, labor-intensive and costly. Preparation of infusate by cold sterilization of dialysis fluid, which is abundantly available, and its immediate (online) use, in contrast, enables a large volume fluid exchange in a cost-effective manner. Recent developments aimed at more hygienic and user-friendly online systems with increased operational flexibility. As a result the novel ONLINEplus system does not only provide online prepared infusate for convective dialysis therapy, but also for priming and rinsing of the extracorporeal blood circuit, for intradialytic bolus administration, and for re-infusion of patients' blood as well. Production of infusate from potentially impure dialysis fluid containing endotoxins and other pyrogens raises severe concerns of affecting the patients' well-being. To assess its safety, the online system was challenged with microbially contaminated dialysis fluid. Despite high levels of microbial counts (7.5 x 104 +/- 105 CFU/ml), endotoxin concentration (14.1 +/- 7.7 IU/ml and 9.265 +/- 3.000 IU/ml, as measured turbidimetrically and chromogenically, respectively) and cytokine-inducing activity (20,827 +/- 3,082 pg IL-1Ra/Mio WBC), we failed to detect contaminants in the final infusate during a 5 week laboratory testing period. In addition, infusate samples complied consistently with the European Pharmacopeia test for sterility. The present online system is comprehensive, operates user-friendly, and provides microbiologically safe infusate in large quantities. In this way, both patients and dialysis staff will benefit from improved dialysis therapy and reduced treatment-related labor burden, respectively. Moreover, convective dialysis modalities will become less expensive.

Can sterile and pyrogen-free on-line substitution fluid be routinely delivered? A multicentric study on the microbiological safety of on-line haemodiafiltration.

BACKGROUND: Microbial contamination is characterized not only by the presence of bacteria, but also by high concentrations of biologically active by-products. They are potentially able to cross ultrafiltration and dialysis membranes and stimulate immunocompetent blood cells to synthesize cytokines. In turn, cytokine induction causes acute symptoms and has been incriminated in the long-term complications of haemodialysis patients. Infusion of large volumes of substitution fluids following ultrafiltration of microbially contaminated dialysis fluids may place patients on on-line therapies at particular risk. METHODS: In this study we evaluated 30 machines with a two-stage ultrafiltration system in routine clinical haemodiafiltration settings in six centres for 6 months. Microbiological safety was assessed monthly and at the last use of the filters by determining microbial counts, endotoxin concentration and cytokine-inducing activity. RESULTS: No pyrogenic episodes were observed during the study period. Double-filtration of standard dialysis fluid (range, <1-895 cfu/ml, 0.0028-4.6822 IU/ml) resulted in sterile substitution fluids with endotoxin concentrations well below the Ph.Eur. standard for haemofiltration solutions (range, 0.0014-0.0281 vs 0.25 IU/ml). Moreover, they did not differ from commercial haemofiltration solutions and depyrogenated saline. Likewise, there was no difference in the cytokine-inducing activity between the solutions tested. The high microbiological quality of the ultrafiltered dialysis fluid, which was in the same range as substitution fluid, translates into both the absence of cytokine induction by dialyser back-transport and a redundant safety mode of the on-line system by a second filtration step. CONCLUSION: On-line HDF treatment can routinely be provided with ultra-pure dialysis fluids and sterile substitution fluids at pyrogen-free levels. The online preparation of substitution fluids thus can be considered microbiologically safe.

Microspheres based detoxification system: in vitro study and mathematical estimation of filter performance.

Because of the closed plasma (secondary) circuit in the Microspheres based Detoxification System (MDS), a convective blood purification system, the same amount of filtrated plasma is backfiltrated into the blood circuit. Therefore, there is no direct way to determine the ultrafiltrate production rate, which is an important factor of efficiency. The only possible way to estimate the filtration properties of the filter is to consider pressure values. In this study the pressure distribution in the filter was investigated in vitro. To explain the results and to calculate inaccessible parameters, a mathematical model was established which also considered the asymmetric behaviour of the filter membrane. The result was a linear pressure gradient, agreement with the measurements was reasonably good (calculated primary pressure loss differs <13% from measured value when using mean measured filter resistance as model parameter). Linear pressure distribution offers the possibility of easily calculating the filtration length, a parameter which can be used to estimate the filter condition. The comparison between calculated filtration and backfiltration rates offers an instrument of control for these values.

In vitro cell culture systems as the basis for an extracorporeal blood purification strategy in multiorgan failure treatment.

Multiorgan failure (MOF) based on septic processes is very common but prognostically an extremely severe disease that has to be treated exclusively under intensive care conditions. Extracorporeal blood purification (ECBP) using specific and efficient systems such as the microspheres based detoxification system (MDS) (Artif Organs 1996;20:420) could improve significantly the situation of MOF in terms of the efficient removal of endotoxins as well as key mediators such as tumor necrosis factor alpha (TNF alpha). The purpose of the study was to test the effectiveness of endotoxin and cytokine removal to blunt cellular response. In terms of the in vitro principle methodology, isolated peripheral blood mononuclear cells (PBMC) were incubated with endotoxins and a selective endotoxin adsorbent, which was added at various times (immediately or 30, 60, 120, 240, or 360 min) after the onset of incubation. TNF alpha release of monocytes was measured following a standard procedure after 20 h. Human TNF alpha was incubated with cultured human endothelial cells with and without a specific TNF alpha adsorbent (polyclonal antibodies coated on polystyrene particles). The results showed that after the initial addition of endotoxins, the activation of monocytes can be stopped within 120 min by addition of endotoxin adsorbents. In addition, specific TNF alpha adsorbents are able to prevent intercellular adhesion molecule 1 (ICAM-1) expression of endothelial cells, therefore avoiding activation of endothelial cells. In conclusion, cell culture models are suitable to simulate cell interaction in MOF. Specific adsorbents are able to reduce or block pathophysiologically relevant cell interactions, and the time frame for effective ECBP seems to be very short, and therefore, efficiency must be high.

Fractionated plasma separation and adsorption system: a novel system for blood purification to remove albumin bound substances.

The removal of albumin bound substances has gained increasing interest in different diseases, especially in acute and chronic liver disease. Therefore, a new system, the fractionated plasma separation and adsorption (FPSA) system, was developed based on combined membrane and adsorbent blood purification techniques. The most important contribution to the FPSA system was the development of a new polysulfone hollow-fiber filter, which is characterized by a sieving coefficient of 0.89 for human serum albumin (HSA) but only of 0.17 for fibrinogen, and 0 (zero) for IgM immunoglobulins. Using a closed filtrate circuit connected to the new polysulfone filter which integrates 1 or 2 adsorption columns and also a high flux dialyzer adapted to a dialysis machine, the FPSA system opens excellent possibilities for the relatively specific removal of albumin bound substances from the blood such as albumin bound bilirubin or even tryptophan. In comparison to other systems (for example, the Molecular Adsorbent Recirculating System [MARS] and albumin dialysis systems), the FPSA system enables much higher elimination of strongly bound albumin substances. The first clinical investigations have recently started based on a modified dialysis machine designed with all necessary safety measures.

Rotary cell culture system (RCCS): a new method for cultivating hepatocytes on microcarriers.

The Rotary Cell Culture System (RCCS) is a new technology for growing anchorage dependent or suspension cells in the laboratory. The RCCS is a horizontally rotated, bubble free disposable culture vessel with diffusion gas exchange. The system provides a reproducible, complex 3D in vitro culture system with large cell masses. During cell growing the rotation speed can be adjusted to compensate for increased sedimentation rates. The unique environment of low shear forces, high mass transfer, and microgravity, provides very good cultivating conditions for many cell types, cell aggregates or tissue particles in a standard tissue culture laboratory. The system enables to culture HepG2 cells on Cytodex 3 microcarriers (mcs) to high densities. We inoculated 2 x 10(5)/ml HepG2 cells and 200 mg Cytodex 3 mcs in 50 ml Williams E medium (incl. 10% FCS) allowing them to attach to the mcs in the rotating vessel (rotation rate 14-20 rpm). HepG2 cells readily attached to the mcs while the vessel was rotating. Attachment of HepG2 to the mcs was about 50% after 24 hrs and 100 % within 48 hrs. After 72 hrs of rotary culturing small aggregates of Hep G2 on mcs were built. HepG2 cells and the aggregates rotated with the vessel and did not settle within the vessel or collide with the wall of the vessel. We conclude that this new RCCS is an excellent technology for culturing HepG2 cells on Cytodex 3 mcs. The system is easy to handle and enables to culture anchorage dependent cells to high densities in a short period.

Monocyte activation and humoral immune response to endotoxins in patients receiving on-line hemodiafiltration therapy.

With the on-line preparation of substitution fluid, an easy-to-operate and cost-effective alternative to conventional hemodiafiltration (HDF) has been realized. The continuous filtration of dialysis fluid, furthermore, allows high volumes of exchange. Microbial contamination and subsequently endotoxins, however, may be present in dialysis fluid, and thus the microbiological safety has become a pivotal issue. In this clinical study we evaluated the safety of the Fresenius Medical Care on-line HDF system which is based on a two-stage filtration of dialysis fluid with upstream DIASAFE and downstream on-line HDF filter. During the three-month study period we failed to detect germs or endotoxins in the substitution fluid. Augmented plasma interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) concentrations were found neither during the intradialytic period nor when pre-session values at study begin and study end were compared. In addition, changes in the anti-endotoxin core antibody levels and soluble CD14 (sCD14) concentration, or pyrogenic episodes were not observed. On-line HDF with DIASAFE and on-line HDF filter thus represents a safe treatment modality by effectively depleting dialysis fluid of cytokine-inducing substances.