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Role of contact system activation in hemodialyzer

carpinteyrosnn

9/5/2013 4:35:00 PM


Role of contact system activation in hemodialyzerDepartment of Nephrology and Clinical Immunology, IZKF "Biomat.," and Institute of Biometry, University Hospital RWTH Aachen, <a href=http://www.karocchio.eu/hollisterfrance/>hollister... Aachen, GermanyReceived 13 July 2000; Revised 23 May 2001; Accepted 4 June 2001.Top of pageAbstract.Background The contact system is generally believed to be the main trigger of the coagulation cascade during extracorporeal circulation. However, the extent of contact activation, its role for intradialytic thrombin generation as well as the influence of different dialyzer membranes have not been well established.Methods In a novel fullscale ex vivo recirculation dialysis model, we investigated the thrombogenicity of three widely used hemodialyzers (Cuprophan Renak RA15U, Polysulfone F6HPS and AN69XT <a href="http://www.cai-catanzaro.it/bottegaveneta/"... bottega veneta imitazioni</a> Nephral 200). The activation of the contact system was evaluated using a newly developed ELISA for factor XIIaC1inhibitor complexes. Additionally, we determined free FXIIa (ELISA), thrombinantithrombin (TAT) complexes, platelet factor 4 (PF4), complement activation (C5a), granulocyte elastase and blood cell counts. The findings in blood from normal volunteers were compared with factor XIIdeficient blood.Results With normal blood AN69 exhibited the highest <a href="http://www.postzegels-poststukken.eu/woolrichjassen/"&g... jassen sale</a> thrombogenicity in comparison to Cuprophan and Polysulfone, as assessed by TAT generation and platelet consumption. AN69 caused a rapid increase of the FXIIaC1inhibitor complexes and of free FXIIa. Despite significant TAT generation with <a href="http://www.photo2video.co.uk/longchamp/">longchamps... Cuprophan and Polysulfone free FXIIa remained unchanged and the FXIIaC1inhibitor complexes stayed below the detection limit. With factor XIIdeficient blood Polysulfone exhibited the same TAT generation, whereas the thrombogenicity of AN69 was greatly reduced.Conclusions Our data challenge the common assumption that activation of the contact system with generation of FXIIa is the main trigger for coagulation and thrombus formation in hemodialysis. In the past, research on the hemocompatibility of dialysis treatment has mainly focused on the complement system and leukocytes. Compared to these data, which have led to a classification of hemodialyzer membranes as more or less "biocompatible"1,2,3, studies evaluating dialyzer thrombogenicity are relatively infrequent and, particularly in vivo, have yielded equivocal results4,5,6,7,8,9. Consequently, no widely accepted dialyzer classification with regard to thrombogenicity is available.Various experimental systems for preclinical membrane testing have been developed10. In many studies blood was incubated under static conditions with dialyzer membrane flat sheets or fragments11,12. Other studies employed downscaled flow systems designed as singlepass ex vivo models with short bloodmaterial contact times13,14,15,16. Both approaches suffer from specific problems: first, since coagulation and platelet activation <a href="http://www.karocchio.eu/hollisterfrance/">hollister... are surface and flowdependent properties, geometry and flow characteristics should reproduce the clinical situation as closely as possible10. Second, single pass as opposed to recirculation systems are relatively insensitive to detect activation products of the humoral or cellular coagulation system, as these cannot accumulate10. Moreover, anticoagulant dosage should mirror the clinical <a href=http://www.photo2video.co.uk/longchamp/>longchamps... situation17. Finally, given the considerable interindividual variability of the response to prothrombotic stimuli and anticoagulants, blood from the same donor should be used when different dialyzers are evaluated in parallel10,16.The first aim of the present study, therefore, was to establish a standardized fullscale ex vivo recirculation dialysis model that allows the simultaneous assessment of the thrombogenicity of two different hemodialyzers. Using this system, our second aim was to gain more insights into the mechanisms by which extracorporeal circulation through different hemodialyzers activates the cellular and humoral coagulation system.Contact phase activation is thought to be the main trigger of the coagulation cascade in extracorporeal circulation like hemodialysis17,18,19,20. Its components, that is, the circulating plasma proteins prekallikrein, high molecular weight kininogen, coagulation factor XI (FXI) and the central zymogen Hageman factor (FXII) rapidly form complexes bound to foreign surfaces, in particular those with negative charge. Autoactivation of FXII then generates trace amounts of the active serine protease FXIIa, which cleaves prekallikrein to kallikrein, thereby initiating a reciprocal FXII activation loop as well as activation of the intrinsic coagulation pathway through the generation of FXIa. Kallikrein converts surfacebound FXIIa by limited proteolysis into alpha and betaFXIIa. The latter retains the proteolytically active site of FXIIa while losing the surfacebinding domain. Released into the fluid phase, betaFXIIa is rapidly bound to and inactivated by potent inhibitors including C1inhibitor and antithrombinforming 1:1 proteaseinhibitor complexes. In normal plasma C1inhibitor accounts for more than 2/3 of the inhibitory potential21,22,23.The extent of the contact system activation and its role in thrombin generation during hemodialysis, as well as membrane differences have not been established clearly. Various in vitro and in vivo investigations suggest that FXII and the contact system may be activated during hemodialysis. Surface bound and supernatant FXIIa activity was detected in static systems using Cuprophan and AN69 membranes incubated with purified FXII, but not with plasma11,12. In vivo, FXIIa generation was demonstrated during hemodialysis in some24 but not all studies25. Further studies noted increased bradykinin and kallikrein activity, that is, indirect markers of contact activation, during the first ten minutes of treatment in the dialyzer effluent26,27, whereas others were not able to demonstrate significant changes28,29.HemodialyzersFor the in vitro recirculation experiments, three commercially available dialyzers matched for intradevice blood volume were used. These dialyzers, designated based on the type of dialyzer membrane, included: (1) "AN69" (polyacrylonitrile, AN69 Nephral 300, 81 mL blood volume, 1.3 m2 membrane surface; Hospal Medizintechnik, N Germany), (2) "Polysulfone" (Hemoflow F6HPS, 82 mL blood volume, 1.3 m2 membrane surface; Fresenius Medical Care, Darmstadt, Germany) and (3) "Cuprophan" (Renak RA15U, 75 mL blood volume, 1.5 m2 membrane surface; Kawasumi <a href="http://www.photo2video.co.uk/abercrombie/">a... and fitch uk</a> Laboratories Europe, D Germany).Blood collectionBlood was collected in the university blood bank from apparently healthy registered blood donors, taken from the blood donor database (N = 13, mean age 36 years, range 23 to 51, 8 males, 5 females), who had not taken any medication for at least two weeks. Each donor volunteered for only one experiment comparing two hemodialyzers in parallel. Two similar polyvinylchloride blood transfer bags (Biotrans, Dreieich, Germany) were prefilled with 25.5 mL heparin solution each, then 230 mL blood were drawn into each bag to yield a final heparin concentration of 0.7 IU/mL blood (blood/heparin solution vol/vol 9:1). The total blood volume collected from each volunteer (460 mL) corresponded to a standard blood donation. Pooled normal human plasma for the FXIIC1inhibitor complex ELISA standard curve was obtained from freshly drawn, citrateanticoagulated blood (0.11 mol/L sodium citrate, vol/vol 9:1) of six additional healthy volunteers registered as regular blood donors. It consists of two parallel running circuits with each circuit containing one conventional roller pump (Medizintechnische Systeme Schweinfurt, Schweinfurt, Germany), one of three different types of commercially available sterile dialyzer modules (types see above) and a specially adapted sterilized tubing system (polyvinylchloride tubing 4.3 6.8, RB3NDG; Modenplast Medical FRL, Italy; volume 70 mL) without air traps (Meise Medizintechnik, Schalksm Germany). Each circuit contained approximately 250 mL of anticoagulated whole blood (0.7 IU heparin/mL) obtained from one donor, circulating for 120 minutes at 37 with a blood flow rate of 200 mL/min. Identical experimental conditions in both circuits were ensured by weighing the blood bags before and after blood filling and a standardized circuit filling procedure. To prevent ultrafiltration, the dialysate compartment was filled with sterile Ringer's solution and sealed. Each <a href=http://www.cai-catanzaro.it/bottegaveneta... bottega veneta imitazioni</a> donor gave blood for <a href=http://www.photo2video.co.uk/abercrombie/>a... and fitch uk</a> one experiment. In each experiment two of the three different dialyzers were tested in parallel with blood from one donor (Cuprophan vs. PS, N = 5; PS vs. AN69, N = 8).Full figure and legend (43K)Before starting the experiments, the dialyzers and tubing systems were filled with Ringer's solution to remove air and to avoid bloodair interfaces, placed into a preheated box, rinsed with 2 liters prewarmed Ringer's solution and allowed to equilibrate at 37 Within 30 minutes after collection, the blood was filled into the experimental setup by carefully replacing the electrolyte solution. A standardized filling procedure produced a similar hemodilution effect in each experiment. To detect a potential preactivation of the coagulation system, control blood samples were taken from the transfer bags immediately before the start of the experiment (0 min). At several time points thereafter (5, 20, 60 and 120 min), additional blood samples were taken from the two circuits, dispensed in different tubes with anticoagulants <Sarstedt>Monovetten containing ethylenediaminetetraacetic acid (EDTA), citrate or, as platelet stabilizer, CTAD (citrate, theophylline, adenosine, dipyridamole), Sarstedt, N Germany] and cooled in an ice bath until further processing. EDTA blood specimens for whole blood counts were kept at room temperature and analyzed on a Coulter MAX hematology analyzer (Coulter Electronics, Krefeld, Germany). During each experiment a total volume of 35 mL blood (7 mL at each time point) was collected from each circuit (14% of circuit blood content). The removed volume was not replaced by air or electrolyte solution.Citrate or EDTA anticoagulated plateletpoor plasma (PPP) was obtained by centrifugation for 12 minutes at 2000 g and 4 Blood samples for platelet factor 4 (PF4) measurement were collected in CTAD and centrifuged for 30 minutes at 2000 g and 4 as recommended by the kit manufacturer. All plasma samples were stored in aliquots at 70 until analysis.Enzymelinked immunosorbent assay (ELISA) for the determination of Factor XIIaC1inhibitor complexMicrotiter plates (NUNC, Roskilde, Denmark) were coated overnight at 4 with a polyclonal factor XII antibody (50 L per well, 10 g/mL in sodium carbonate: 0.05 mol/L NaHCO3, 0.05 mol/L Na2CO3, pH 9.5). After coating and between all incubation steps, the plates were washed four times with washing buffer (2.7 mmol KCl, 1.5 mmol KH2PO4, 137 mmol NaCl, 6.5 mmol Na2HPO4, 0.1% Tween 20, pH 7.4). Nonspecific binding sites in the solid phase were blocked with 2% BSA in washing buffer (60 min at 37 The wells were then incubated for 60 minutes at 37 with aliquots (50 L) of plasma samples, standards or controls, diluted 1/8 with washing buffer. In the next step, the biotinylated antibody (50 L per well, 0.5 g/mL in washing buffer) was pipetted into the wells and incubated for 60 minutes at 37 Antigenantibody complexes were detected by addition of streptavidinconjugated horseradish peroxidase (50 L per well, 1/1000 in washing buffer; incubation 60 min at 37 followed by a chromogenic reagent (ophenylendiamin, 50 L per well, 0.4 mg/mL, 0.05 mol/L phosphate citrate buffer, pH 5.0, containing 4 L 30% H2O2 per 10 mL). After 30 minutes at 37 the reaction was stopped by the addition of 50 L 2N HCl. Color development was quantified at 492 nm using an automatic ELISA plate reader (SLT Labinstruments Deutschland GmbH, Crailsheim, Germany).Standards were prepared by activating citrateanticoagulated pooled plateletpoor plasma at 37 with PBS (vol/vol <a href=http://www.postzegels-poststukken.eu/woolrichjassen/&g... jassen sale</a> 1:1) containing 20 mg Kaolin per mL. After a 60minute incubation time the Kaolin was removed by centrifugation at 3000 g, 21 for 10 minutes. The activated plasma was stored in small aliquots at 70 The amount of FXIIaC1inhibitor complex in the activated plasma was arbitrarily set as 100% activation. Standards and controls were prepared by mixing activated plasma with pool plasma. The addition of pool plasma (diluted 1:1 with PBS) to Kaolinactivated plasma caused no further FXIIC1inhibitor complex formation. Prior to use, the standards and controls were diluted again with washing buffer (vol/vol 1:4) in order to achieve a total dilution of 1:8.Further assaysIn addition to the FXIIaC1inhibitor complex ELISA described above, factor XII activation also was assessed by determining the level of free factor XIIa using a commercially available FXIIaELISA (Shield Diagnostics, Dundee, UK).