Ex vivo evaluation of the blood compatibility of mixed matrix haemodialysis membranes.

The patients with end stage kidney disease need haemodialysis therapies, using an artificial kidney. Nevertheless, the current therapies cannot remove a broad range of uremic toxins compared to the natural kidney. Adsorption therapies, using sorbent-based columns, can improve the clearance of uremic toxins, but the sorbent particles often require polymeric coatings to improve their haemocompatibility leading to mass transfer limitations and to lowering of their performance. Earlier, we have developed a dual layer Mixed Matrix fiber Membrane (MMM) based on polyethersulfone/polyvinylpyrrolidone (PES/PVP) polymer blends. There, the sorbent activated carbon particles are embedded in the outer membrane layer for achieving higher removal whereas the inner blood contacting selective membrane layer should achieve optimal blood compatibility. In this work, we evaluate in detail the haemocompatibility of the MMM following the norm ISO 10993-4. We study two generations of MMM having different dimensions and transport characteristics; one with low flux and no albumin leakage and another with high flux but some albumin leakage. The results are compared to those of home-made PES/PVP single layer hollow fiber and to various control fibers already applied in the clinic. Our results show that the low flux MMM successfully avoids contact of blood with the activated carbon and has good haemocompatibility, comparable to membranes currently used in the clinic. STATEMENT OF SIGNIFICANCE: Haemodialysis is a life-sustaining extracorporeal treatment for renal disease, however a broad range of uremic toxins cannot still be removed. In our previous works we showed that a double layer Mixed Matrix Membrane (MMM) composed of polyethersulfone/polyvinylpyrrolidone and activated carbon can achieve higher removal of uremic toxics compared to commercial haemodialysers. In this work we evaluate the haemocompatibility profile of the MMM in order to facilitate its clinical implementation. The lumen particle-free layer of the MMM successfully avoids the contact of blood with the poorly blood-compatible activated carbon. Moreover, thanks to the high amount of polyvinylpyrrolidone and to the smoothness of the lumen layer, the MMM has very good haemocompatibility, comparable to membranes currently used in the clinic.

Haemodiafiltration at increased plasma ionic strength for improved protein-bound toxin removal.

AIM: Protein-bound uraemic toxin accumulation causes uraemia-associated cardiovascular morbidity. Enhancing the plasma ionic strength releases toxins from protein binding and makes them available for removal during dialysis. This concept was implemented through high sodium concentrations ([Na+ ]) in the substituate of pre-dilution haemodiafiltration at increased plasma ionic strength (HDF-IPIS). METHODS: Ex vivo HDF-IPIS with blood tested increasing [Na+ ] to demonstrate efficacy and haemocompatibility. Haemocompatibility was further assessed in sheep using two different HDF-IPIS set-ups and [Na+ ] between 350 and 600 mmol L-1 . Safety and efficacy of para-cresyl sulphate (pCS) and indoxyl sulphate (IS) removal was further investigated in a randomized clinical pilot trial comparing HDF-IPIS to HD and HDF. RESULTS: Compared to [Na+ ] of 150 mmol L-1 , ex vivo HDF-IPIS at 500 mmol L-1 demonstrated up to 50% higher IS removal. Haemolysis in sheep was low even at [Na+ ] of 600 mmol L-1 (free Hb 0.016 ± 0.001 g dL-1 ). In patients, compared to HD, a [Na+ ] of 240 mmol L-1 in HDF-IPIS resulted in 40% greater reduction (48.7 ± 23.6 vs. 67.8 ± 7.9%; P = 0.013) in free IS. Compared to HD and HDF (23.0 ± 14.8 and 25.4 ± 10.5 mL min-1 ), the dialytic clearance of free IS was 31.6 ± 12.8 mL min-1 (P = 0.017) in HDF-IPIS, but [Na+ ] in arterial blood increased from 132 ± 2 to 136 ± 3 mmol L-1 (0 vs. 240 min; P < 0.001). CONCLUSION: HDF-IPIS is technically and clinically feasible. More effective HDF-IPIS requires higher temporary plasma [Na+ ], but dialysate [Na+ ] has to be appropriately adapted to avoid sodium accumulation.

Guanidinylations of albumin decreased binding capacity of hydrophobic metabolites.

AIM: As post-translational modifications of proteins may have an impact on the pathogenesis of diseases such as atherosclerosis, diabetes mellitus and chronic kidney disease (CKD), post-translational modifications are currently gaining increasing interest. In this study, a comprehensive method for analysis of these post-translational modifications is established for the clinical diagnostic routine. METHODS: Here, we analysed albumin - the most abundant plasma protein in human - isolated from patients with CKD and healthy controls by chromatographic steps and identified by MALDI mass spectrometry. Post-translational modifications of albumin were identified after digestion by analysing mass signal shifts of albumin peptides using pertinent mass databases. RESULTS: Albumin isolated from plasma of patients with CKD but not from healthy control subjects was specifically post-translationally modified by guanidinylation of lysines at positions 249, 468, 548, 565 and 588. After identification of guanidinylations as post-translational modifications of albumin isolated from patients with CKD, these modifications were quantified by mass spectrometry demonstrating a significant increase in the corresponding mass signal intensities in patients with CKD compared to healthy controls. The relative amount of guanidinylation of lysine at position 468 in patients with CKD was determined as 63 ± 32% (N = 3). Subsequently, we characterized the pathophysiological impact of the post-translational guanidinylation on the binding capacity of albumin for representative hydrophobic metabolic waste products. In vitro guanidinylation of albumin from healthy control subjects caused a decreased binding capacity of albumin in a time-dependent manner. Binding of indoxyl sulphate (protein-bound fraction) decreased from 82 ± 1% of not post-translationally modified albumin to 56 ± 1% after in vitro guanidinylation (P < 0.01), whereas the binding of tryptophan decreased from 20 to 4%. These results are in accordance with the binding of indoxyl sulphate to albumin from healthy control subjects and patients with CKD (88 ± 3 vs. 74 ± 10, P < 0.01). Thus, in vitro post-translational guanidinylation of albumin had a direct effect on the binding capacity of hydrophobic metabolites such as indoxyl sulphate and tryptophan. CONCLUSION: We used a mass spectrometry-based method for the characterization of post-translational modification and demonstrated the pathophysiological impact of a representative post-translational modification of plasma albumin. The data described in this study may help to elucidate the pathophysiological role of protein modifications.

FGF23 is independently associated with vascular calcification but not bone mineral density in patients at various CKD stages.

SUMMARY: The hormone fibroblast growth factor 23 (FGF23) is involved in mineral homeostasis but may also have a role in vascular calcification and bone mineralization. In a cohort of 142 patients with CKD stages 2-5D, plasma FGF23 was independently associated with aortic calcification but not with pulse wave velocity or bone mineral density. INTRODUCTION: FGF23 is involved in mineral homeostasis but may also have a role in vascular calcification and bone mineralization. Previous studies related to FGF23 and vascular and bone outcomes have been restricted to dialysis patients. The aim of the present study was to establish whether or not plasma FGF23 is associated with aortic and coronary calcification, arterial stiffness, and bone mineral density in patients with early as well as late stages of CKD. METHODS: In a cohort of 142 patients with CKD stages 2-5D, we made routine biochemistry and intact FGF23 determinations, and assessed aortic and coronary calcification, bone mineral density (BMD), and arterial stiffness by multislice spiral computed tomography and automated pulse wave velocity (PWV). RESULTS: Plasma intact FGF23 levels were elevated in CKD patients; the elevation preceded that of serum phosphate in early-stage CKD. Patients with elevated FGF23 levels had higher aortic and coronary calcification scores than patients with lower FGF23 levels. Multivariate linear regression analysis indicated that only age (p < 0.001) and FGF23 (p = 0.008) were independently associated with aortic calcification score. Plasma FGF23 was neither associated with PWV nor with BMD. CONCLUSION: Our data suggest that plasma FGF23 is an independent biomarker of vascular calcification in patients with various CKD stages including early stages. The association between vascular calcification and FGF23 levels appears to be independent of BMD. It remains to be seen whether this association is independent of bone turnover and bone mass.

New miniaturized hollow-fiber bioreactor for in vivo like cell culture, cell expansion, and production of cell-derived products.

We have developed a miniaturized hollow-fiber bioreactor system for mammalian cell culture with a volume of 1 mL. Cell and medium compartments of the bioreactor are separated by a semipermeable membrane, and oxygenation of the cell compartment is accomplished using an oxygenation membrane. As a result of the geometry of the transparent housing, cells can be observed by microscopy during culture. The leukemic cell lines CCRF-CEM, HL-60, and REH were cultivated up to densities of 3.5 x 10(7)/mL without medium change or manipulation of the cells. As shown using CCRF-CEM cells, growth in the bioreactor was strongly influenced and could be controlled by the medium flow rate. As a consequence, consumption of glucose and generation of lactate varied with flow rate. Depending on the molecular size cutoff of the membranes used, added growth factors such as GM-CSF, as well as factors secreted from the cells, are retained in the cell compartment for up to 1 week. This new miniaturized hollow-fiber bioreactor offers advantages in tissue engineering by continuous nutrient supply for cells in high density, retention of added or autocrine produced factors, and undisturbed long-term culture in a closed system.

Monitoring of cell viability and cell growth in a hollow-fiber bioreactor by use of the dye Alamar Blue.

We describe a method for monitoring cell proliferation in a small-scale hollow-fiber bioreactor (culture volume: 1 ml) by use of the Alamar Blue dye. Alamar Blue is a non-fluorescent compound, which yields a fluorescent product after reduction, e.g. by living cells. In contrast to the MTT-assay, the Alamar Blue assay does not lead to cell death. However, when not removed from the cells, the Alamar Blue dye shows a reversible, time- and concentration-dependent growth inhibition as observed for the leukemic cell lines CCRF-CEM, HL-60 and REH. When applied in the medium compartment of a hollow-fiber bioreactor system, the dye is delivered to the cells across the hollow-fiber membrane, reduced by the cells and released from the cell into the medium compartment back again. Thus, fluorescence intensity can be measured in medium samples reflecting growth of the cells in the cell compartment. This procedure offers several advantages. First, exposure of the cells to the dye can be reduced compared to conventional culture in plates. Second, handling steps are minimized since no sample of the cells needs to be taken for readout. Moreover, for the exchange of medium, a centrifugation step can be avoided and the cells can be cultivated further. Third, the method allows discriminating between cell densities of 10(5), 10(6) and 10(7) of proliferating HL-60 cells cultivated in the cell compartment of the bioreactor. Measurement of fluorescence in the medium compartment is more sensitive compared to glucose or lactate measurement for cell counts below 10(6) cells/ml, in particular. We conclude that the Alamar Blue-assay combined with a hollow-fiber bioreactor offers distinct advantages for the non-invasive monitoring of cell viability and proliferation in a closed system.

Pyrogen retention by highly permeable synthetic membranes during in vitro dialysis.

Pyrogen permeability of the new highly permeable synthetic membrane polyethersulfone (DIAPES) was compared to polysulfone in vitro dialysis experiments with heparinized human donor blood in the blood compartment. After sterile dialysis for 5 min, dialysate was contaminated with a culture filtrate of Pseudomonas aeruginosa using high and moderate challenge doses (Limulus assay reactivity 20,000 EU/ml and 50 EU/ml, respectively). Whole blood samples were separated from the blood compartment during the sterile (5 min) and contaminated (60 min) phases of dialysis and incubated for 6 h at 37 degrees C. Blood samples were lysed, and interleukin-1beta and tumor necrosis factor alpha were measured by specific ELISAs. Moderate dialysate contamination (50 EU/ml) did not induce detectable cytokine production in whole blood. High challenge dose (20,000 EU/ml) induced whole blood interleukin-1beta and tumor necrosis factor alpha production in the blood compartment, which was higher with DIAPES than with polysulfone after 30 min. After 60 min, membrane-dependent differences were no longer detectable. Pyrogen concentrations in the dialysate decreased with time indicating adsorption of cytokine-inducing substances to the dialyzer membrane. Pyrogens adsorbed to dialyzer membranes were resuspended during recirculation of sterile phosphate-buffered saline in the dialysate compartment and retained cytokine-inducing activity as seen from whole blood incubation experiments. DIAPES and polysulfone adsorbed pyrogens in the presence of whole blood. Pyrogen adsorption to the membrane polymer and/or to the protein coat on the membrane prevented the passage of pyrogens in the presence of moderately contaminated dialysate. High grade dialysate contamination caused breakthrough of pyrogens into the blood with DIAPES and polysulfone. In order to reduce the risk of a dialysis-dependent inflammatory response, dialysate of high bacteriological quality (ultrapure dialysate) should be mandatory.

Low-molecular but not high-molecular advanced glycation end products (AGEs) are removed by high-flux dialysis.

BACKGROUND: Patients with end-stage renal disease (ESRD) display very high levels of advanced glycation end products (AGEs). These compounds are suspected to play a pathophysiological role in diabetic nephropathy and late diabetic cardiovascular complications. We investigated to what extent AGE levels can be reduced by high-flux dialysis. PATIENTS AND METHODS: Ten ESRD patients were treated three times each with DIAPES and HF60, two different synthetic, high-flux hemodialysis membranes. The kinetics of AGE removal was studied by fluorescence spectroscopy (excitation 370 nm/ emission 440 nm) and by ELISA of serum samples and the removal of beta2-m was studied by immunonephelometry of plasma samples. Samples were taken during dialysis sessions at t = 0, 30 and 180 min. In addition, molecular weight distribution of AGE products in serum of three patients was analyzed by gel filtration and fluorescence detection. RESULTS: A significant difference could be found when AGE levels in serum of controls (n = 10) were compared with serum AGE levels of ESRD patients (p < 0.01/fluorescence; p < 0.0001/ ELISA). After 3 h of dialysis AGE-related fluorescence in serum decreased by 25.5 +/- 6.8% for HF60 (p < 0.0001) and 24.3 +/- 6.9% tor DIAPES (p < 0.0001). The corresponding decline measured by ELISA was 23.3 +/- 8.9% for HF60 (p < 0.0001) and 26.1 +/- 7.0% for DIAPES (p < 0.0001). Both methods showed no significant differences for both types of dialysis membranes. Gel filtration revealed that the decrease of fluorescence can be attributed to the removal of AGE peptides with a molecular mass < 12 kDa, only. In the high molecular range (> 12 kDa) no removal but hemoconcentration was observed independent of the dialyzer type used. The reduction of beta2-m during 3 hours of dialysis was 61.8 +/- 6.9% for HF60 (p < 0.0001) and 161.7 +/- 7.0% for DIAPES (p < 0.0001). CONCLUSION: Both high-flux dialyzers were equally effective to remove low-molecular AGE products, while AGE-modified proteins of higher molecular weight were only marginally affected. On the basis of our data we suggest the study of molecular mass-dependent uremic toxicity of AGEs and the examination of the influence of other treatment modalities on the level of high-molecular AGEs.

Anaphylactoid reactions during hemodialysis in sheep are ACE inhibitor dose-dependent and mediated by bradykinin.

Anaphylactoid reactions (AR) have been attributed to the generation of bradykinin (BK) when AN69 membranes are used together with angiotensin converting enzyme (ACE) inhibitors during hemodialysis. However, conclusive evidence for the involvement of the BK as the mediator of these AR is still lacking. This study examined the degree of contact activation in an animal model caused by three PAN membranes--AN69, PAN DX, and SPAN- and the effects of different doses of the ACE inhibitor enalapril (ENA) and the BK B2-receptor antagonist icatibant on AR during hemodialysis. Six sheep were dialyzed for one hour with or without ENA pre-treatment using the different membranes in random order. Severe AR were observed only during hemodialysis with AN69 dialyzers together with ENA pre-treatment; the severity of AR increased with the ENA dose. Mild hypotension was noted during hemodialysis with AN69 without ACE inhibition and with PAN DX and 20 mg ENA. Compared to pre-dialysis values, maximum generation of BK after blood passage through the dialyzer was found at five minutes: 73-fold (AN69 without ENA), 161-fold (AN69 with 10 mg ENA), 97-fold (AN69 with 20 mg ENA), 108-fold (AN69 with 30 mg ENA), 154-fold (AN69 with 30 mg ENA and 0.1 mg/kg icatibant), 18-fold (PAN DX without ENA), and 42-fold (PAN DX with 20 mg ENA). Elevated BK levels in arterial blood were detected during hemodialysis with AN69 membranes even without ACE inhibition (2.5-fold); pre-treatment with 20 mg ENA further increased arterial BK concentrations (4-fold). Furthermore, a marked decline of prekallikrein and high molecular weight kininogen concentrations was noted for both AN69 and PAN DX membranes. Anaphylactoid reactions during hemodialysis were completely prevented by icatibant even after pre-treatment with ENA and in the presence of high BK concentrations. Concentrations of prekallikrein, high molecular weight kininogen, and BK remained unchanged and no AR were observed during hemodialysis with SPAN and pre-treatment with 20 mg ENA. Our findings confirm that AR during hemodialysis with the negatively charged AN69 membrane are mediated by BK, since they can be prevented by the BK B2-receptor antagonist icatibant.

Determination of advanced glycation end products in serum by fluorescence spectroscopy and competitive ELISA.

Recent studies suggest that advanced glycation endproducts play an important role in cardiovascular complications of ageing, diabetes and end-stage renal failure. Since highly elevated levels of advanced glycation endproducts are present in serum of patients on maintenance haemodialysis, an accurate and rapid assay for their determination would be useful. This would be particularly valuable for monitoring the removal of advanced glycation endproducts by novel dialysis membranes, as well as the effect of new drugs for the inhibition of their formation. Measurement of advanced glycation endproducts in serum was performed by two competitive ELISAs, using a monoclonal antibody directed against imidazolone, an advanced glycation endproduct formed by the reaction of arginine with 3-deoxyglucosone, and a polyclonal antibody directed against keyhole limpet haemocyanin-advanced glycation endproduct, as well as by quantitative fluorescence spectroscopy. Each of the assays showed significant differences between the controls and the maintenance haemodialysis patients. Advanced glycation endproduct levels determined by each of the ELISAs correlated with total and protein-bound fluorescence, but not with each other, suggesting a variable distribution of advanced glycation endproducts on serum proteins among the maintenance haemodialysis patients.

No generation of bradykinin with a new polyacrylonitrile membrane (SPAN) in haemodialysis patients treated with ACE inhibitors.

BACKGROUND: Anaphylactoid reactions occurring in uraemic patients haemodialysed with polyacrylonitrile haemodialysis (HD) membranes and being treated with ACE inhibitors have been attributed to an excessive generation of bradykinin. METHODS: Here we tested in a prospective trial a new type of polyacrylonitrile membrane (SPAN) with respect to bradykinin generation in nine HD patients receiving either captopril or enalapril. Each patient had three consecutive HD sessions with each of the three tested membranes, high-flux SPAN, high-flux polysulphone (F60) and low-flux Hemophan (GFS Plus 16). RESULTS: No clinical signs of anaphylactoid reactions were observed in any of these patients but the number of patients was relatively small and the duration of exposure to different membranes relatively short. At 5 min after the start of HD session, plasma bradykinin levels were significantly higher in the venous than in the arterial line for all three HD membranes: SPAN, 18.5 +/- 11.9 versus 12.4 +/- 5.3 fmol/ml (P < 0.05); F60, 19.0 +/- 13.8 versus 11.5 +/- 6.5 fmol/ml (P < 0.01); and GFS Plus 16, 39.1 +/- 22.9 versus 15.8 +/- 12.4 fmol/ml (P < 0.005), mean +/- SD respectively. Higher venous line levels were still observed at the 15 and 60 min time points for F60 and GFS Plus 16, but not for SPAN. However, these levels were still insignificant compared to levels measured during episodes of anaphylactic shock from the literature. Plasma histamine and C5a anaphylatoxin levels did not show any increase during HD with SPAN. CONCLUSION: The SPAN membrane did not induce significant bradykinin release in dialysis patients on ACE-inhibitor therapy. It may therefore be used for high-flux dialysis in such patients.

Kinin generation by hemodialysis membranes as a possible cause of anaphylactoid reactions.

1. Severe anaphylactoid reactions have been reported in some patients treated with angiotensin converting enzyme (ACE) inhibitors during hemodialysis with a polyacrylonitrile (PAN) membrane. Generation of bradykinin via contact activation at the negatively charged membrane surface and reduced bradykinin breakdown due to ACE inhibition have been suggested as possible causes. This hypothesis was evaluated in the present study. 2. PAN or cellulose dialyzer membranes were incubated with plasma at different concentrations of ACE inhibitor. The rate and extent of kinin accumulation was dependent on the ACE inhibitor concentration. 3. Bradykinin levels were determined in "historical" plasma samples drawn from patients treated with ACE inhibitor at the onset of anaphylactoid reactions during hemodialysis with PAN dialyzers. The kinin levels were significantly higher (2.4 +/- 0.05 pmol/ml) than in samples from a group of control patients (0.29 +/- 0.02 pmol/ml). 4. Plasma kinin levels were measured in patients who developed anaphylactoid reactions during dialysis with a PAN membrane though not being treated with ACE inhibitor. At the onset of the reaction, kinin levels increased to 2.1 pmol/ml in the line entering the dialyzer and to 10.5 pmol/ml in the line leaving the dialyzer compared to not more than 0.12 pmol/ml upon dialysis with other membranes. 5. These in vitro and in vivo results demonstrate that contact of blood with a polyacrylonitrile membrane leads to the generation of kinins which accumulate if ACE is inhibited. It is very likely that kinin accumulation in the circulation is the cause of anaphylactoid reactions during hemodialysis with PAN membranes in patients treated with ACE inhibitors and, in some cases, in patients not receiving ACE inhibitor medication.

Cellulose-ester as membrane materials for hemodialysis.

The majority of dialysis membranes are fabricated from regenerated unmodified cellulose. This standard type of cellulosic membrane is frequently under attack because of its alleged lack of biocompatibility. Recent developments, however, have proven that a chemical modification of the reactive surface groups of regenerated cellulose, the hydroxyl-groups, limits the complement-activating potential of these materials and thus improves its blood-compatibility. We extended the idea of modifying cellulose for improved blood-compatibility to a series of different cellulose esters. Special focus was directed towards the question whether a variation of the type of substituent and degree of substitution could influence the blood-compatibility pattern of these materials: the analysis of blood-compatibility profiles showed a direct dependency on the type of substituent and the degree of substitution (DS). As an example, it was found that the DS, necessary for a complete reduction of complement activation, decreases with increasing chain lengths of aliphatic substituents. Optimal degrees of substitution are characteristic of the type of substituents and enable us to tailor materials specifically for optimized blood compatibility.

Bradykinin is a mediator of anaphylactoid reactions during hemodialysis with AN69 membranes.

Anaphylactoid reactions (AR) are the most feared complications of hemodialysis. Recently, a high incidence of AR has been reported during dialysis with AN69 membranes in patients treated with ACE inhibitors. Plasma levels of C3a, histamine and bradykinin were measured in 12 patients at the onset of AR during dialysis with AN69. We also investigated bradykinin generation in 10 symptom-free patients dialyzed with four different membranes. None of the 12 patients studied during AR displayed excessive complement activation or histamine release. In contrast, high bradykinin plasma levels (2392 +/- 53 fmol/ml; mean +/- SEM) were observed in all nine patients of whom bradykinin was measured. One patient developed two consecutive episodes of hypersensitivity on AN69 membranes even without taking ACE inhibitors. Bradykinin levels were high in both episodes (5280 and 10467.7 fmol/ml). Furthermore, this patient showed no symptoms and normal bradykinin levels (123.4 fmol/ml) when dialyzed with other membranes. The role of the membrane type in the AR is further substantiated by the observation that AN69 also provoked a significantly higher bradykinin generation (327.6 +/- 18 fmol/ml; mean +/- SEM) during symptom-free sessions compared to other membranes like CuprophanR (5.1 +/- 7.3), HemophanR (17.2 +/- 6.3) and PolysulfoneR (39.7 +/- 6.6). Our findings strongly suggest that bradykinin is the principal mediator of AR during hemodialysis with AN69 membranes. To our knowledge it is the first time that data support the hypothesis of a more general role of bradykinin in shock-like symptoms. Furthermore, bradykinin generation must be regarded as a new marker of biocompatibility of extracorporeal treatments.

Methods for the assessment of neutrophil function during extracorporeal circulation.

Many different assays and test systems have been used to study the effects of biocompatibility on neutrophil function in haemodialysis patients. This review examines the most common and recommends three assays that are widely available, amenable to standardization, and minimally susceptible to artefacts. The recommended assays are neutrophil count, plasma concentrations of the elastase- alpha 1-proteinase inhibitor complex (a measure of neutrophil degranulation), and, the neutrophil oxidative burst response as measured by the reduction of ferricytochrome-c by superoxide or the oxidation of dihydrorhodamine 123 by hydrogen peroxide. This review also discusses the advantages and disadvantages of using whole blood or isolated cell assays and the choice of in-vitro, on-line, or clinical dialysis test systems. While whole blood assays are associated with fewer artefacts than those using isolated cells, specialized equipment is needed to study neutrophils and some questions concerning localization of defects in neutrophil function can only be addressed in isolated cells. For these reasons a role for isolated cell assays continues. For similar reasons there remains a role for in-vitro and on-line test systems, even though clinical dialysis studies are preferred whenever possible.

In vitro biocompatibility evaluation of a heparinizable material (PUPA), based on polyurethane and poly(amido-amine) components.

The biocompatibility of a new heparinizable material based on polyurethane and poly(amido-amine) (PUPA) was evaluated both in the heparinized and non-heparinized forms. The quantity of heparin present on the material was measured using radiolabelled heparin and biological tests. Heparin release in plasma from heparinized PUPA was investigated using in vitro methods. The behaviour of PUPA towards cellular and plasmatic blood components was studied. The influence of sterilization on the cytocompatibility response of both heparinized and non-heparinized PUPA was investigated; gamma-rays were found to be a suitable method of sterilization as no toxic response was noticed.

Hypersensitivity reactions during haemodialysis: the choice of methods and assays.

Our considerations about assays to assess agents and mechanisms involved in hypersensitivity reactions can be summarized as follows. The measurement of total IgE is recommended since it confirms a history of atopy of the patient and is a quick and inexpensive laboratory assay. If, based on the anamnesis, a particular allergen can be suspected to be involved in the clinical reaction a RAST against this compound is highly recommended. In any case of a severe clinical reaction the RAST against ethylene oxide is mandatory to confirm or exclude the involvement of ethylene oxide. The radioallergosorbent test (RAST) should not be used as a screening test for an entirely unknown allergen. The determination of histamine in plasma samples can be applied for specific scientific questions but cannot be recommended as a routine test with first priority. In contrast, the measurement of bradykinin should be established as soon as possible as a routine test for all new materials which are developed for application in the extracorporeal circuit. An initiative of the haemodialysis community is recommended to assure the commercial availability of a reliable bradykinin immunoassay.

Activation of human neutrophils by mastoparan. Reorganization of the cytoskeleton, formation of phosphatidylinositol 3,4,5-trisphosphate, secretion up-regulation of complement receptor type 3 and superoxide anion production are stimulated by mastoparan.

In human neutrophils, mastoparan induced rapid F-actin polymerization which was followed by a slow and sustained depolymerization to below the initial F-actin content. Incubation of neutrophils with pertussis toxin inhibited mastoparan-stimulated actin polymerization; however it did not prevent sustained depolymerization of F-actin. Analyses of phospholipids performed in parallel revealed that mastoparan stimulated rapid formation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and consumption of phosphatidylinositol 4,5-bisphosphate (PIP2). Pertussis toxin treatment blocked mastoparan-induced formation of PIP3. Furthermore, mastoparan stimulated the release of N-acetylglucosaminidase from primary granules. Cytochalasin B enhanced mastoparan-stimulated secretion. Mastoparan triggered superoxide radical production in a cytochalasin B-sensitive manner and induced complement type 3 receptor (CR3) up-regulation.