Complement Activation During Hemodialysis: Laboratory Evaluation of Hemodialyzers

A laboratory method that facilitates delineation of the complement-activating characteristics of various dialyzers under defined conditions has been developed. Results obtained by circulating reconstituted human serum through these devices and measuring time-dependent production of both C3a and C5a antigens are entirely consistent with previous clinical observations. For example, the complement-activating potential of dialyzer membranes could be described as high (cuprammonium cellulose), moderate (cellulose acetate), or low (polycarbonate or polyacrylonitrile). Furthermore, these techniques provided the opportunity to identify membrane characteristics that are not readily defined by clinical studies alone. Specifically, membranes that transported and absorbed C5a antigen were readily identified by these methods. Additionally, laboratory evaluation provided the unique ability to define the efficiency of complement activation taking place on the membrane surface. Results of these investigations are compatible with a hypothetical model that not only describes the properties of a typical dialyzer membrane but may be generally applicable to other biomaterials as well.     

Leukopenia, Hypoxemia, and Complement Activation During a Single Hemoperfusion

White blood cell count, acid-base balance, PO2, and complement function in five uremic patients undergoing a single hemoperfusion employing activated charcoal coated with methacrylate were studied. After 20 min on hemoperfusion, a marked leukopenia [ranging from 6,080 +/- 526 to 3,740 +/- 1,124 (p less than 0.02)] and hypoxemia [ranging from 106 +/- 13.8 to 80.2 +/- 11.9 mm Hg (p less than 0.02)] were observed. At the same time, total hemolytic complement decreased from 135 +/- 15.7 to 123 +/- 14.7 U/ml (p less than 0.001) and alternative pathway activity from 38.1 +/- 5.1 to 33.1 +/- 6.7 U/ml (p less than 0.005). C3 and B cleavage fragments were detected in the samples tested, thus demonstrating the activation of the complement alternative pathway. After 60 min, the different parameters tended to increase but did not reach the baseline levels. A direct correlation between the degree of leukopenia and the reduction of PO2 throughout the hemoperfusion period was found. pH PCO2, and HCO-3 did not change throughout the hemoperfusion period. The results demonstrate that complement activation, leukopenia, and hypoxemia occur during hemoperfusion.     

Complement Activation Produced by Biomaterials

The complement-activating potential of biomaterials may be defined by appropriate application of C3a and C5a anaphylatoxin radioimmunoassays. Studies performed with hemodialysis membranes demonstrate that blood contact with these model biomaterials results in complement activation that may be ascribed to specific properties of the material surface. Further delineation of these chemical and physical properties may permit design of biocompatible materials.     

Role of Dialyzer Contaminants in the Allergic Epiphenomena of Hemodialysis

Abstract: Cuprophan hollow-fiber dialyzers contain contaminants including 1,2,3-propanetriol, carbohydrates, Limulus amebocyte lysate-reactive material, and particulates. In a clinical study, the role of these substances in the allergic-type response seen in some hemodialysis patients was examined. Patients were dialyzed three times per week for 6-week intervals with each of four dialyzer preparations designed to vary the burden of contaminants presented to the patient. Predialysis eosinophil counts and serum immunoglobulin (Ig) E levels were obtained weekly. White cell and platelet counts and plasma C3a and C5a levels were measured during dialysis for each dialyzer preparation. Dialyzer preparation had no effect on predialysis eosinophil counts or IgE levels. All patients demonstrated transient leukopenia and complement activation during dialysis, the magnitudes of which were unaffected by the type of dialyzer preparation. At the levels found in the dialyzers studied, it was questioned whether water-soluble extractables or particulates play any role in the allergic epiphenomena of hemodialysis.     

Complement Activation During Low-Density Lipoprotein Apheresis

Complement system activation was investigated in two girls with familial homozygous hypercholesterolemia undergoing two monthly sessions on LA15 or LA40 (Kaneka liposorber). We determined blood levels of C3c and C3a, leukocyte counts, and plasma levels of C3c and C3a in the extracorporeal circulation device at the start of the sessions and 15 and either 60 or 120 min into them. Sequential eluates were collected from LA40 at the end of the sessions (0.5M NaCl, 1M hydroxylamine). Anaphylatoxin C3a increased throughout, especially with LA40. As previously reported, C3a was trapped in the dextran column but was noticeably present in efferent plasma. Besides many proteins, nonnative complement fragments bearing C3a and C3d antigens were detected in almost all the eluates, suggesting possible in situ complement activation. Practically, complement activation induced by the first filter is a risk; long-term side effects may arise from this extracorporeal circulation device.     

Effect of Dialyzer Reprocessing Methods on Complement Activation and Hemodialyzer-Related Symptoms

The effects of different dialyzer processing methods and of reuse on complement activation and dialyzer-related symptoms were studied in 96 maintenance hemodialysis patients. New dialyzers were either unprocessed (Group 1) or machine-washed with bleach and stored in formaldehyde (Group 2). Reused dialyzers were manually cleansed using the combination of bleach and formaldehyde (Group 3), or machine-washed in formaldehyde (Group 4) or peracetic acid (Group 5). Prewashed new dialyzers (Group 2) were associated with greater complement activation during dialysis when compared with unprocessed, new dialyzers (Group 1) (p less than 0.05). Reused, unbleached but formaldehyde-treated or peracetic acid-treated dialyzers (Groups 4 and 5) were associated with reduced complement activation (p less than 0.05). Complement activation was not reduced when bleach was used for reprocessing (Group 3). The percentage of patients without symptoms during dialysis was significantly greater with reused dialyzers than with new dialyzers (Groups 3 through 5 versus Groups 1 and 2; 39 versus 25%; p = 0.035). The severity of total symptoms correlated significantly (p = 0.0004) with complement activation. Our results suggest that total symptoms during dialysis are correlated with the degree of complement activation. However, trends in the data pertaining to chest pain suggest that factors other than complement activation may be important in the pathogenesis of some dialyzer-related symptoms.     

A New Model for Evaluation of Biocompatibility: Combined Determination of Neoepitopes in Blood and on Artificial Surfaces Demonstrates Reduced Complement Activation by Immobilization of Heparin

Abstract: An in vitro technique was developed for assessment of the biocompatibility of materials for use in clinical applications. Artificial materials were exposed to blood, and the resulting complement activation was quantified both in the plasma and on the material surface by enzyme immunoassays based on monoclonal antibodies specific for neoepitopes exposed in complement activation products. Several materials were evaluated, and the effect of surface modifications, including end–point immobilized heparin, was studied. The results revealed widely varying complement activation properties of the different materials and confirmed that heparin markedly improves biocompatibility. The present method is superior to analysis limited to either the fluid phase or solid phase since certain materials adsorb activation products (exemplified by Tecoflex) whereas others do not although activation was evident from fluid–phase assay (silicone). Furthermore, direct determination of activation–specific neoepitopes on the surface represents an improvement compared with previously described methods for detection of adsorbed components.     

Changes in Platelet, Granulocyte, and Complement Activation During Cardiopulmonary Bypass Using Heparin-coated Equipment

Abstract: The effects of heparin-coated cardiopulmonary bypass (CPB) systems on platelet, granulocyte, and complement activation were investigated during cardiopulmonary bypass. Thirty patients underwent coronary artery bypass surgery with a heparin-coated (Carmeda Bio-Active Surface, CBAS, Medtronic, U.S.A.) CPB system (HC group, n = 10), a heparin-coated oxygenator and uncoated CPB circuit (HO group, n = 10), or an uncoated system (UC group, n = 10). In the HO group, plasma C3a (1667 ± 632 ng/ml) and C4a (1088 ± 319 ng/ml) concentrations were significantly (p < 0.05) lower than in the UC group (2846 ± 1045 ng/ml and 1494 ± 480 ng/ml, respectively) 10 min after the administration of protamine, but there were no significant differences in the platelet or granulocyte counts. In the HC group, granulocyte elastase concentrations 120 min after the onset of CPB (365 ± 177 μg/L) and 10 min after the administration of protamine (676 ± 314 μg/L) were significantly (p < 0.05) lower than in the other 2 groups (820 ± 341 and 893 ± 303 μg/L and 1365 ± 595 and 1,258 ± 622 μg/L). In addition, the increase in the plasma C3a concentration in the HC group 60 (p < 0.05) and 120 min after the onset of CPB (p < 0.05) was significantly less than in the other 2 groups. The C3a and C4a concentrations 10 min after the administration of protamine were significantly (p < 0.005 and p < 0.05) less in the HC group than in the UC group. Platelet counts 10 min after the administration of protamine were significantly higher (p < 0.05) and plasma β-throm-boglobulin concentrations during CPB were significantly lower in the HC group than in the other 2 groups 5 (p < 0.05), 60, and 120 min (p < 0.005) after the onset of CPB. Postoperative blood loss during the first 12 h in the HC group was significantly (p < 0.05) less than that in the UC group. The heparin-coated oxygenator and uncoated CPB circuit reduced complement activation but demonstrated no significant effects on the platelet and granulocyte systems. However, the heparin-coated CPB circuit (with all components making blood contact) reduced platelet, granulocyte, and complement activation and significantly reduced postoperative blood loss. Therefore, heparin coating of CPB systems improves biocompatibility.     

Recirculation Measures with Urea and Mannitol During Hemodialysis

Abstract: A simple, accurate, and reproducible method of measuring recirculation in grafts during hemodialysis is essential for improving the efficiency of dialysis. In our studies, plasma samples for plasma urea nitrogen (PUN) were taken from the arterial line of the dialyzer at blood flows (A) of 200, 300, and 400 ml/min, preceded by a 5–min period of equilibration, and at 15 s and 2 min after turning the flow down to 100 ml/min (S), the latter serving as systemic samples. Recirculation was calculated as (S – A)/(S – V). Total blood flow (Q_b) through the grafts was measured by color Doppler ultrasound. We found a significant, inverse relationship between recirculation and total flow through the graft at dialyzer Q_b of 400 but not 300 or 200 ml/min. The magnitude and prevalence of recirculation was always greater when the 2 min sample was used as S compared to the 15 s sample and as dialyzer Q_b increased. As a qualitative, urea–independent measure of recirculation, we assayed the appearance of mannitol in the arterial line in blood drawn 15 s after initiating a mannitol push into the venous line. Blood obtained just prior to the mannitol push was used as the zero blank. Thirteen of 18 patients had a measurable, but low, level of mannitol, 5 did not, and 2 had inconsistent results in studies done on separate days. We conclude that the majority of patients receiving chronic hemodialysis have a low degree of recirculation and that methods relying on urea must be suspected of exaggerating the true degree of recirculation.     

Sodium Modeling During Hemodialysis: A New Approach

Sodium volume modeling during hemodialysis encounters several difficulties. First, the actual sodium distribution volume is the extracellular water, whereas the ultrafiltration flow reflects the variation of total body water. Thus, a two-pool model must be considered. This will complicate the model by increasing the number of parameters and boundary conditions. An alternative is to consider the total body water as the apparent distribution volume of loaded or removed sodium, which leads to a single-pool model. Second, convective sodium transfer induced by ultrafiltration is not negligible compared with diffusive sodium transfer. Therefore, sodium transfer modeling must simultaneously take into account the diffusive and the convective part, with the coupling part related to both processes. Third, the Donnan effect due to nondiffusible anionic plasma proteins modifies the sodium transfer through the membrane. Adequate sodium volume modeling should be a compromise between oversimplification, resulting in discrepancies between calculated values and experimental data, and overcomplexity, involving a great number of parameters and boundary conditions, which leads to a model unsuitable for clinical application. A single-pool model is proposed with only one parameter that is estimated during the first period of the hemodialysis session.     

Filling a Dialysis Circuit with Albumin Does Not Prevent Platelet Activation During Hemodialysis: In Vivo Study

Abstract: The aim of our prospective clinical study was to evaluate whether filling a dialysis circuit with albumin before hemodialysis (HD) can prevent platelet activation during the procedure. Eight patients with chronic HD participated in the study, each dialyzed first with albumin and a week later without it. All other parameters of the HD procedure were unchanged (cellulose acetate hollow fiber dialyzer, blood flow of 300 ml/min, and low dose heparin). Before HD with albumin, 6. 7% human albumin in saline was recirculated in the dialysis circuit for 10 min at a flow rate of 100 ml/min, and infused into the patient. We found a significant increase in the beta–thromboglobu lin levels during both procedures. We found no difference in plasma coagulation system activation measured by fibrinopeptide A concentration. Neither was there any difference in the macroscopic antithrombotic activity assessed at the end of HD by measuring the volume of clots in the arterial and venous bubble trap and by counting the number of clotted fibers in the dialyzer. It seemed that filling the dialysis circuit, which had a cellulose acetate dialyzer, with human albumin did not improve its thrombogenicity.     

Influence of Hemodialysis on Plasma Chemotactic Activity and the Chemotactic Responsiveness of Polymorphonuclear Neutrophils

Thirty patients with chronic renal failure on maintenance hemodialysis (HD) were studied. Plasma chemotactic activity was estimated using the "under agarose" chemotaxis assay during the first 2 h of HD. It was found that in the fifth minute of HD, patients' plasma became chemotactic, reaching the maximum activity at the tenth minute. The chemotactic activity appearance correlated significantly with the decline in the number of the peripheral neutrophils. Patients' neutrophils, after a single passage through the cellophane coil of the dialyzer, revealed significant impairment of directed migration toward both complementary and bacterial chemoattractants. Moreover, the chemotactic properties of neutrophils obtained from dialyzed patients before HD were significantly lower than had been estimated in 15 nondialyzed patients with chronic renal failure. The results confirm HD-induced complement activation and might explain the mechanisms of the increased susceptibility of dialyzed patients to bacterial infections.     

Plate, Coil, and Hollow-Fiber Cuprammonium Cellulose Dialyzers: Discrepancy Between Incidence of Anaphylactic Reactions and Degree of Complement Activation

During the past 10 years, the incidence of severe anaphylactic reactions during dialysis [type A first-use syndrome (FUS)] at our center has been much lower when using cuprammonium cellulose plate (CC-P) dialyzers (0/37, 750 dialyses) or coil (CC-C) dialyzers (0/32, 500) than when using cuprammonium cellulose hollow-fiber (CC-F) dialyzers (8/21,022 dialyses, p less than 0.005 by Chi-square). To determine if the difference in type A FUS incidence between the three dialyzer types could be explained by differences in complement activation, we compared plasma concentrations of C3a des-arginine (des arg) in patients undergoing dialysis with these three varieties of dialyzers. Plasma C3a des arg values increased markedly in the dialyzer outflow blood with the three dialyzer configurations. The levels were similar with the dialyzer types when results were corrected for membrane surface area. Also, the degree of leukopenia was not markedly different with the three dialyzer types. Our findings suggest that complement activation per unit surface area is similar during dialysis with plate, coil, and hollow-fiber cuprammonium cellulose dialyzers. The lack of correlation between the degree of complement activation and the incidence of type A FUS suggests that membrane-induced complement activation is not of primary importance to type A dialyzer hypersensitivity reactions.     

Plasma Cytokine Levels and Coagulation and Complement Activation During Use of the Extracorporeal Liver Assist Device in Acute Liver Failure

Multiple organ failure frequently occurs in patients with acute liver failure, and this has been associated with increased cytokine production. Treatment by hemoperfusion with an extracorporeal liver assist device (ELAD) containing human liver-derived cells was performed in 12 patients with acute liver failure. Over the first 6 h, there were significant increases in plasma tumor necrosis factor alpha (TNFα; from 114 ± 54 pg/ml [mean ± SEM] to 236 ± 161 pg/ml, p < 0.05) and interleukin (IL)-6 (260 ± 121 pg/ml to 445 ± 149 pg/ml, p < 0.05) but not in interferon gamma (IFNγ). A similar pattern with a small peak increase was observed for complement C5b-9 complex. Plasma C-reactive protein (CRP) and thrombin antithrombin (TAT) III complex showed small peaks after 24 h of ELAD hemoperfusion. No such changes were seen in 12 control patients with acute liver failure who were treated with intensive care alone. These transitory effects, without changes in blood pressure, are likely to be due to the contact of the blood with the dialyzer membrane. There was no evidence of the clearance of cytokines by the ELAD.     

Levomepromazine Elimination in Patients During Active and Sham Hemodialysis

Abstract: The effect of active and sham dialysis on the plasma concentration of levomepromazine was studied in seven schizophrenic patients undergoing hemodialysis treatment in a double-blind crossover study. Samples of blood were collected before, after 2 h and 5 h of treatment, and once a week during the treatment program. The plasma concentrations decreased during both active and sham dialysis. The data indicate that there was no significant difference in the elimination of the drug between active and sham hemodialysis.     

Hemodynamic Changes During Acetate and Bicarbonate Hemodialysis

In a crossover, double-blind comparison, circulatory changes induced by hemodialysis with bicarbonate versus acetate dialysate were evaluated at the first exposure as well as after 2 weeks of acclimatization to each dialysate. Hemodialysis with bicarbonate dialysate resulted in only minor changes in blood pressure and left ventricular (LV) function as assessed by M-mode echocardiography. In contrast, the first exposure to acetate resulted in significant decreases in systolic (30 mm Hg) and diastolic (17 mm Hg) blood pressure as well as in LV end-diastolic and end-systolic dimensions (5-6 mm) and a rise in ejection fraction. After acclimatization, tolerance developed for the arterial vasodilatory effects of acetate, but not for the venous vasodilatory effect (persistent decrease in LV end-diastolic dimension). These results indicate that some of the circulatory changes induced by hemodialysis may be related more to the acetate infused than to fluid losses or relative autonomic insufficiency.     

Biocompatibility in Hemodialysis: Clinical Relevance in 1995

Abstract: Hemodialysis therapy for end-stage renal disease is still empirical even after more than 30 years of experience. Although long-term survival can now be assured in selected patients, clinical results tend to be disappointing. Hemodialysis therapy needs to be improved. Zealots of the biocompatibility school believe that this improvement will come from reducing undesirable consequences of blood membrane interaction, particularly complement activation. However, there is controversy over the clinical meaningfulness of biocompatibility when exclusively related to blood membrane interactions. Another dimension needs to be added, namely ultrapure di-alysate to avoid subclinical chronic effects of activation of the cytokine cascade by bacterial fragments present in dialysate. While the pathogenesis of acute anaphylactoid reactions are understood and largely preventable, the relation of the chronic syndromes such as amyloidosis to the use of a particular membrane remain unproven. Prospective studies that will occupy at least a decade will be necessary to decide these issues.