1 Nephrology Department, Lapeyronie University Hospital, Montpellier, France, 2 Georg Haas Dialysis Center, Giessen, Germany, 3 Nephrology and Dialysis Unit, S. M. Annunziata Hospital, Florence, Italy, 4 Department of Renal Medicine, Lister Hospital, Stevenage, UK, 5 Dialysis Center Villingen-Schwenningen, Germany, 6 Fresenius Medical Care, Bad Homburg, Germany and 7 Center for Biomedical Technology, Danube University Krems, Austria
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Abstract |
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Methods. The microbiological safety of a novel on-line system, ONLINEplusTM, 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/106 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/106 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.
Keywords: chronic inflammation; cytokine-inducing substances; endotoxins; interleukin-1 receptor antagonist; on-line haemodiafiltration
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Introduction |
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However, on-line preparation of infusate by the dialysis machine may be associated with enhanced risk of pyrogenic reactions if not operated with proper hygienic awareness. On a more subtle level, there is increasing evidence of harm resulting from recurrent exposure to moderate levels of pyrogens during dialysis. Microbial contamination of dialysis water and dialysate, which is common in dialysis centres [46], results in by-products that are capable of crossing both high- and low-flux dialysis membranes. Blood exposure to these substances activates leukocyte synthesis of pro-inflammatory cytokines, including interleukin-1 (IL-1) and tumour necrosis factor (TNF
), as well as counter-inflammatory mediators such as interleukin-1 receptor antagonist (IL-1Ra) [69]. In addition to provoking acute clinical symptoms, dialysis-related cytokine induction may contribute to subclinical chronic inflammation, which has been incriminated in malnutrition, cardiovascular disease, immunodeficiency, and hyporesponsiveness to erythropoeitin therapy [1014]. Furthermore, inflammatory response markers in recent studies were predictive of hospitalization and mortality in chronic haemodialysis patients [1317].
On-line dialysis modalities are characterized by the preparation of large volumes of infusate by multi-stage ultrafiltration (cold sterilization) of dialysis fluid. Therefore, administration of infusate may affect both acute and long-term patient health unless removal of endotoxins and other cytokine-inducing substances is complete. ONLINEplusTM, a novel on-line system, was designed to ensure infusate preparation at the highest microbiological standards. Although the infusate may be used as substitution for saline, it may also be employed as a supplement for saline before, during, and at the termination of a dialysis session. Using this technology, the extracorporeal blood circuit can be quickly and automatically filled (primed) and rinsed before dialysis, as well as emptied afterwards. In addition, boli of infusate can easily be injected into the extracorporeal circuit when needed, which obviates the need for pre-packed saline.
Laboratory investigations of microbiological safety have demonstrated the great capacity of this system for removing microorganisms and microbially derived pyrogens from dialysis fluid [18]. In the present study, we assessed the performance of the system in a multicentre clinical setting. Specifically, we addressed the impact of on-line HDF on inflammatory response in dialysis patients by analysing ex vivo IL-1Ra production as a sensitive marker of chronic inflammation.
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Subjects and methods |
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Dialysis fluid downstream from the first filter stage enters the dialyser (for HDF, or is by-passed for HF); part of the stream is subjected to cross-flow filtration in the second filter in order to produce infusate. The infusate stream, in turn, can be connected via disposable tubes with the arterial patient connector of the blood line (for priming, rinsing, re-infusion) and with the arterial or venous bubble catcher for pre-dilutional and post-dilutional HDF/HF, respectively. Materials and design of both the hydraulic system and the connectors are optimized to prevent microbial colonization.
Citric acid or peracetic acid (Citrosteril® at 84°C, and Puristeril® 340 at 37°C, respectively; Fresenius Medical Care) was used to clean and disinfect the machine as well as the infusate circuit with both ultrafilters after each dialysis session. Citric acid disinfection was combined with a limited number of bleach cycles (11 cycles per filter life; Sporotal® 100, Fresenius Medical Care, at 37°C) in two centres.
Patients and treatment
Sixteen stable end-stage renal disease (ESRD) patients (three female, 13 male), aged 59 years (range 3876), received post-dilutional HDF with ONLINEplusTM for 3 months. ESRD was due to glomerulonephritis, polycystic kidney disease, diabetes mellitus, interstitial nephropathy, and ischaemic kidney disease. Previous renal replacement therapy included on-line HDF for 22 months (range 148) with 2008 and 4008 dialysis machines (Fresenius Medical Care), high-flux polysulfone membranes, using 35 and 1620 l of infusate in pre- and post-dilutional modes, respectively. Close microbiological monitoring of dialysis water was implemented in all study centres.
None of the patients suffered from chronic infection, malignancy or autoimmune disease, or received medication that might interfere with the immune system. Twelve healthy individuals (four female, eight male, aged 47 years (range 3068)) served as a control group.
On-line HDF was performed for 4 h (range 2.55) with non-reprocessed HF80 dialysers (Fresenius Medical Care) and standard bicarbonate dialysate. Blood flow as well as dialysate and infusate flow rates were set at 300400, 500800, and 70110 ml/min, respectively. The overall fluid exchange volume in excess of the patient's ultrafiltration prescription averaged 19 l (range 1627) per session.
Dialysis fluid and infusate sampling
Dialysis fluid samples were taken upstream from the first filter stage (inlet dialysis fluid) as well as downstream from the first filter stage before entering the dialyser (ultrapure dialysis fluid). Infusate was collected from the disposable infusate line before entering the venous bubble catcher. Samples taken bi-weekly during the first session after the weekend shut-down period were immediately cultured for microbial growth or collected in non-pyrogen-adsorbing polystyrene tubes and frozen (-18°C) for the measurement of Limulus amoebocyte lysate (LAL) reactivity and cytokine-inducing activity. Microbial contamination of infusate was additionally checked in one centre by interposing a membrane filter (0.45 µm nominal pore size; Millipore, Bedfort, US) in the infusate line during the entire dialysis session.
Determination of microbial counts
Dialysis fluid and infusate samples (0.11 ml) were processed in duplicate on tryptic soy agar (TSA) or Reasoner's nutrient poor agar (R2A) (both from Difco Laboratories, Detroit, US) and cultured for 3 days at 37°C, or 7 days at ambient temperature (2022°C), respectively. Filter membranes interposed in the infusate line were aseptically removed from the filter holder after treatment and cultured on R2A medium as described above.
Determination of endotoxin concentration
Samples were analysed for endotoxins (lipopolysaccharides, LPS) using a kinetic chromogenic LAL assay (Chromogenix, Mölndal, Sweden) with a detection threshold of 0.009 IU/ml. All determinations were performed in duplicate by a single person. The absence of sample-derived interfering factors was checked prior to each measurement. Isotonic saline autoclaved for 3 h served as endotoxin-free control.
Measurement of cytokine-inducing activity
Blood drawn from healthy subjects into pyrogen-free heparinized tubes (Chromogenix, Mölndal, Sweden) was pooled and incubated with dialysis fluid and infusate samples at 37°C for 24 h (50 µl blood+200 µl sample). Following centrifugation, IL-1Ra was quantified in the supernatant by ELISA (Biosource, Nivelles, Belgium) and normalized to white blood cell (WBC) counts. All incubations were performed in duplicate by a single person. Baseline values were established with isotonic saline autoclaved for 3 h. Production of IL-1Ra was significant at 20 pg/ml LPS from Pseudomonas aeruginosa (Sigma, St Louis, MO, US), and was strongly associated with the generation of IL-1ß and TNF, as measured by ELISA (Biosource, Nivelles, Belgium) (Figure 1
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Measurement of the cytokine induction
Blood samples were drawn from the afferent arterial blood line before and at the end of a dialysis session. Following collection into pyrogen-free heparinized tubes (Chromogenix, Mölndal, Sweden), blood was fractionated and analysed for cytokine induction. Briefly, 1 ml of blood was added to 4 ml of RPMI 1640 medium (Biochrom, Berlin, Germany) containing either no endotoxin (spontaneous cytokine production), low levels of endotoxin (final concentration 100 ng/ml LPS from Salmonella abortus equi (Sigma, St Louis, MO, US); LPS-stimulated cytokine production), or high levels of endotoxin (final concentration 10 µg/ml LPS; maximum cytokine production or cytokine production capacity). All samples were mixed and incubated at 37°C for 24 h. Following gentle resuspension of blood cells, the samples were centrifuged and the supernatant was analysed for IL-1Ra by ELISA (Biosource, Nivelles, Belgium).
Statistical analysis
Data presented as means±SEM were first analysed for normality of distribution using the KolmogorovSmirnov test for normality. When comparing two groups, normally distributed data were analysed by Student's t-tests for unpaired and paired samples, and non-normally distributed data by MannWhitney's U-test and the Wilcoxon test for paired samples as appropriate. The level of significance was set at P<0.05.
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Results |
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The determination of endotoxins in inlet dialysis fluid revealed low-grade contamination with LAL reactivity levels ranging from 0.002 to 0.279 IU/ml (0.020±0.008 vs control, 0.0043±0.0004 IU/ml, P<0.001). Cytokine-inducing activity, assessed by IL-1Ra production during whole blood culture, showed slight but non-significant elevations in inlet dialysis fluid when compared with pyrogen-free control (187±20 vs 148±12 pg IL-1Ra/106 WBC, P=0.08). Ultrafiltration of inlet dialysis fluid resulted in ultrapure dialysis fluid, which did not differ from pyrogen-free control with respect to both LAL reactivity and IL-1Ra-inducing activity (Table 1). Similarly, no differences were found between infusate and control for either parameter (Table 1
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Discussion |
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Microbiological tests including 1-week incubation on R2A and 3-day incubation on TSA media showed low-grade contamination in dialysis fluid upstream from the on-line system. Samples analysed in parallel showed a similar number of colony forming units with no significant differences between the methods. Compared with recent multicentre reports on microbiological quality of dialysis water and dialysate [46], the present findings revealed a high purity of dialysis fluid, resulting from hygienic awareness, regular cleaning and disinfection, and adequate microbiological monitoring of water delivery systems.
The ultrafiltration of dialysis fluid produced an ultrapure dialysis fluid that was depleted in microorganisms, endotoxins, and overall IL-1Ra-inducing activity. Retention capacity of filters was not affected by the cleaning and disinfection regimen and did not decline over the study period. Instead, a pyrogen-free dialysis fluid was consistently produced, and there were no differences between new filters at study entry and life-time depleted after 3 months of use. These results confirm previous investigations demonstrating the retention capability of this on-line system [18]. Although Weber et al. [18] challenged ultrafilters with several log-dilutions of dialysis fluid heavily contaminated with various Pseudomonas species; they did not detect bacteria, endotoxins, or cytokine-inducing activity in ultrapure dialysis fluid. However, a limited retention capacity was indicated by cytokine-inducing substances that crossed the upstream filter during the least diluted challenge, particularly in combination with hypochlorite disinfection. These findings prompted the authors to recommend minimal standards for dialysis water and concentrates to avoid exhausting the ultrafilter retention capacity. They additionally proposed the use of validated agents for disinfection without excessive bleaching [18].
Compliance with these standards permits safe on-line dialysis as demonstrated in the present study. The infusate, obtained from double-stage filtration of dialysis fluid did not differ from pyrogen-free saline control, and therefore contained no microorganisms, endotoxins or other cytokine-inducing material. In addition, there were no differences between the negative control and ultrapure dialysis fluid (downstream of the first filter) demonstrating the redundant operation of the downstream filter stage with respect for all parameters examined. Membrane integrity was demonstrated by regular pressure holding tests and careful in vitro examination following filter replacement. Thus, the on-line infusate is comparable to pharmaceutical quality standards; it is both sterile and pyrogen-free when operated according to manufacturer's specifications because of filter redundancy and a validated filter manufacture process.
A microbiologically pure infusate should cause no inflammatory burden for the patient despite large volumes of substituted fluid. In addition, ultrapure dialysis fluid minimizes the risk of back-diffusion mediated back-transport of cytokine-inducing substances in the dialyser and haemofilter. To assess the inflammatory status in our patients we have measured the activation of immunocompetent cells by ex vivo cytokine production in whole blood culture. Whole blood, rather than isolated blood cell cultures, closely mimicks the natural cell environment and takes advantage of interactions between various leukocyte subsets. In addition, whole blood cultures require less blood, are easy to perform and to standardize, and are associated with few preparation artefacts. Although IL-1Ra, by blocking IL-1 activities, has anti-inflammatory rather than pro-inflammatory properties, it can be considered a superior cytokine marker for leukocyte cell activation [19]. In vivo and invitro studies have shown that IL-1Ra is largely produced and released by leukocytes, mostly neutrophils and monocytes, and appears in higher concentrations than pro-inflammatory species [6,7,1925]. Most importantly, IL-1Ra generation correlates with pro-inflammatory markers of cell activation [6,7,2125], which is consistent with our finding that IL-1Ra production is highly associated with the generation of IL-1ß and TNF.
Using whole blood cultures, we confirmed previous reports examining chronic inflammation and leukocyte activation in ESRD patients, by showing levels of IL-1ß, TNF, and IL-1Ra production compared with healthy subjects [19,24,26,27]. Whole blood co-incubated with minute amounts of LPS showed greater IL-1Ra production in ESRD patients than in healthy individuals. Similarly, the ratio between low-dose endotoxin-stimulated and maximum IL-1Ra production was much higher in ESRD patients than in controls. These findings are in agreement with a recent report by Vaslaki and co-workers [24] who found significantly higher levels of IL-1Ra and TNF
in culture supernatants of LPS-stimulated whole blood from ESRD patients than from healthy subjects. Persistent leukocyte activation, however, does not seem to be accompanied by marked down-regulation of cytokine synthesis. Instead, the pre-dialysis capacity of circulating leukocytes to produce IL-1Ra was not impaired in our patients compared with healthy individuals. These observations are consistent with recent reports showing normal IL-1ß and TNF
production during high-dose LPS co-incubation of whole blood from on-line HDF patients [24,28].
Analysis of the spontaneous IL-1Ra generation over the 3-month study period suggests that on-line HDF does not markedly contribute to the inflammatory response of dialysis patients. Pre-session IL-1Ra production remained essentially unchanged after 36 treatments and an average of approximately 700 l of infusate administered per patient. We also failed to observe elevated IL-1Ra levels in supernatants of unstimulated post-dialysis whole blood cultures compared with pre-dialysis cultures, indicating that leukocytes were not significantly stimulated during the treatment. Similar results were reported by Pizzarelli et al. [2] who found no intradialytic production of IL-1ß, TNF, or IL-1Ra by peripheral blood mononuclear cells (PBMC) from on-line HDF patients. Intradialytic leukocyte stimulation with concomitant IL-1ß, TNF
, and IL-1Ra synthesis has been observed during standard HDF and HD in some studies [8,25,29,30], but not in others [24,31]. Differences in ESRD patient populations and dialysis biocompatibility, including the microbiological dialysate quality, as well as methodological differences in cytokine production determination may account for these discrepancies.
The chronic activation of leukocytes from on-line HDF patients in the present study may be related more to the metabolic and immunologic disturbances of uraemia than to the dialysis procedure per se. In fact, levels of IL-1ß, TNF, and IL-1Ra production in previous studies were shown to be enhanced in uraemic patients before entering dialysis when compared with healthy controls [19,26]. However, cytokine induction is complex and various cytokine-inducing factors such as complement activation, shear stress, cell-membrane contact, uraemic metabolites, and pyrogens may act synergistically. Cytokine expression involves a strongly regulated sequence beginning with transcription (production of mRNA) followed by translation into protein and intracellular protein targeting. Minor stimulation may result in so-called pre-stimulated or primed cells, showing degradation of cytokine mRNA rather than translation [32]. We hypothesized that low-dose pyrogen exposure during dialysis may result in transcription rather than translation of IL-1Ra, as previously shown for anaphylatoxin C5a as a stimulus for IL-1ß and TNF
gene expression in PBMC [32]. Similarly, bioincompatible HD therapy was associated with augmented transcription but impaired translation of IL-6, suggesting that cytokine gene expression and protein synthesis may be independently modulated by the dialysis procedure [31]. In order to assess cytokine induction on the transcriptional level, we incubated whole blood following low-dose endotoxin exposure. LPS may serve as a secondary stimulus to augment translation of cytokine mRNA, inducing augmented cytokine production in primed compared with unstimulated cells, as was shown with IL-1ß [32]. Although IL-1Ra appears to be translated more readily and by a greater variety of stimuli than IL-1ß, recent evidence indicates that steady-state IL-1Ra mRNA levels are similar to those of IL-1ß mRNA [21]. In addition, various stimuli including LPS and TNF
induced IL-1Ra and IL-1ß transcription in circulating neutrophils with similar kinetics [21]. Furthermore IL-1Ra-inducing factors may stabilize IL-1Ra transcripts and significantly prolong their half-life [33]. Taken together, these studies indicate that pyrogens may be effective in generating IL-1Ra transcripts during dialysis. Incubation with LPS from whole blood culture should therefore translate transcripts into final IL-1Ra protein. In our study, however, we failed to observe a post-dialytic increase in LPS-stimulated IL-1Ra generation, indicating that intradialytic leukocyte activation is unlikely to occur on the transcriptional level. In addition, although pre-dialysis LPS-stimulated IL-1Ra production was higher than in healthy control subjects it did not increase further during the 3-month study period.
The lack of intradialytic leukocyte stimulation may have resulted from an altered population of circulating WBCs. Cytokine production may have been attenuated because of prevailing immature or functionally impaired leukocytes following sequestration of activated cells into the lung vasculature. Schindler and co-workers [34] demonstrated that mononuclear cells, once activated to express cytokine genes, did not return to the circulation within the time frame of the dialysis session. In our study, whole blood was cultured with 10 µg/ml of endotoxins to overcome varying LPS-binding capacity, such as with LPS-binding protein, bactericidal or permeability-increasing protein, and soluble CD14 in plasma from different donors. The endotoxins also maximally activated leukocytes for IL-1Ra production. Our findings of stable WBC counts and an unaffected post-dialysis IL-1Ra production capacity suggest that leukocytes did not significantly exit from the vascular compartment following treatment-related cell activation. In addition, they confirmed that spontaneous and LPS-stimulated ex vivo IL-1Ra generation, as performed in this study, are valid indicators of intradialytic leukocyte activation.
We conclude that operation of on-line systems with hygienic awareness, adequate dialysis water quality, regular replacement of ultrafilters and validated cleaning and disinfection procedures permits a safe production of infusate, as demonstrated for a novel on-line system in a multicentre investigation. With this technology infusate can be used for substitution, for example on-line HDF, rinsing of the extracorporeal blood circuit, and bolus application without significant impact on cytokine induction and chronic inflammatory response.
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Acknowledgments |
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Notes |
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References |
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