Complement Activation Induced by Different Tubings Used for Cardiopulmonary Bypass

____________ _ Complement activation induced by latex, polyvinylchloride, and silicone tubings for extracorporeal circulation was studied in 2 in vitro situations: Fresh whole human blood mixed with priming solution was circulated through 1 00 em tubing lengths for 1 hour, and small tubing pieces were incubated in fresh human serum for 7 hours. Activation of C3 and of the terminal pathway (C5-C9) was assessed in enzyme immuno assays. In both situations complement activation was significantly higher (p<O.OS) than in controls. During incubation, C3 activation started later with silicone, but increased to similar levels as with polyvinylchloride after 7 hours. Latex was a significantly more potent C3 activator at 7 hours than the other materials (p<0.05), whereas polyvinylchloride induced most terminal pathway activation (p<O.Ol ). When blood was pumped through intact tubing, complement activation was similar with all materials. The differences found during incubation were apparently overshadowed by other factors in the dynamic situation. The study thus gives no evidence for preferring any of the tested tubing materials to reduce complement activation during in vivo extracorporeal circulation, even if the tubing does contribute to overall activation. Introduction ____________ _ It is well known that complement cascade activation during cardiopulmonary bypass (CPB) may have several Address correspondence to: Vibeke Videm, MD. Institute for Experimental Medical Research, Uni1·ersity of Oslo. Ullevaal Hospital. N-0407 Oslo 4. Norway. I Institute for Experimental Medical Research, University of Oslo, 2 Department of Surgery, Ullevaal Hospital 3 Institute of Immunology and Rheumatology, The National Hospital, Oslo, Norway. Volume 21, Number 1, Spring 1989 adverse effects ( 1-4): The activation product C3a acts as a cardiodepressant (5). Another activation product. C5a, may be a pathogenic factor in development of pulmonary capillary leak and non-pressure edema ( 1,6) and of multi-organ failure (7) after CPB. Deposition of the terminal complement complex ITCC) on erythrocytes may be partly responsible for the hemolysis observed during CPB (8). Complement is activated via the alternative pathway when blood comes into contact with the artificial polymers used in the CPB circuit (2.9). Investigations of membranes for dialysis have shown that the degree of complement activation in this situation of blood-polymer contact depends on the type of material used ( 10). The differences in ability to activate complement is related to the binding capacity for the activation product C3b and various regulatory proteins on the surface of the specific material (II). The aim of the present study was to evaluate complement activation induced by different tubing used for CPB. Tubing segments were first tested in a dynamic situation. i.e. by circulating blood through the tubing using a roller pump, and no significant differences were found. Thereafter, we tested the tubing in a static situation by incubation of small pieces in serum. now observing significant differences between the various materials. Materials and Methods ________ _ Experiments with intact tubing Five brands of tubing for extracorporeal circulation were tested, each with 6 parallel experiments: Bypass 65 PVC (a), Latex (b), Rehau Silicone (c), R.X. Silicone I d), and Tygon (e). Each piece of tubing (length: 100 em. a Bentley Laboratories B. V., Uden, Holland b Kent Latex Products, Kent, Ohio, USA c Raumedic Rehau, Rehau, West Germany d Dow Corning, Midland, Michigan, USA e Norton, Akron, Ohio, USA The Journal of Extra-Corporeal Technology 29 inner diameter: 13 mm, wall thickness: 2.4 mm) was formed in a circle by means of a connector including a bleed port. Four pieces of tubing randomly chosen from the 5 brands were delivered in sterile sets (a) where all tubing circles could be filled simultaneously from a common cardiotomy reservoir (f). Fresh whole human blood from informed volunteer donors was collected in standard CPD-adenin bags (g). For each tubing set, I unit of blood with 2,500 IU of Heparin was filled into the reservoir. A baseline sample was drawn before a priming solution containing 125 ml Dextran 60 mg/ml in NaCI, 125 ml Ringer's acetate, 125 ml Glucose 50 mg/ml, 125 ml Mannitol 150 mg/ml, and 50 ml sodium hydrogen carbonate 500 mmol/1 was added. and the tubing was filled. The blood/prime mixture was circulated through the tubing at 4 !/min for I hour using 4 different roller pumps (h). Test samples were obtained after 15, 30. 45. and 60 minutes. Controls containing blood mixed with Ringer's acetate (n = 8) or priming solution (n = 6) were set up in glass tubes. All samples were drawn into glass tubes containing ethylenediamine tetraacetic acid (EDTA) and kept on ice until they were centrifuged shortly thereafter. The plasma was stored at 70°C. Incubation experiments Fresh human serum was prepared from informed voluntary blood donors. The temperature was maintained at 4°C immediately after collection. "'EDT A-serum" (10 mmoll I) for controls was prepared from the same serum. The following 4 tubing brands were tested in 6 parallel experiments: Bypass 65 PVC. Latex. R.X. Silicone. and Tygon. The tubing was cut into pieces of approximately 2 x 5 mm. and similar amounts of pieces (determined by relative specific weights) of the various types of tubing were incubated in fresh serum at room temperature. Controls consisted of fresh serum without tubing pieces (n=6), tubing pieces in "EDTA-serum" (n=4. i.e. one of each brand) and "EDT A-serum" without tubing pieces (n = 2). Samples for examination of complement activation were collected after 4 and 7 hours. Analyses of samples In the dynamic test C3 activation was measured in a double antibody enzyme-linked immunosorbent assayELISA (12). Briefly, the plates were coated with a rat monoclonal antibody (Clone 9) specific for a C3 "g" neoepitope expressed after activation at the level of iC3b. but not on native C3. This antibody was a kind gift from f Polystan, Copenhagen, Denmark g Laboratories Travenol, La Chatre, France h Cambro, Horten, Norway Dako, Copenhagen. Denmark 30 The Journal of Extra-Corporeal Technology Prof. Peter J. Lachmann. A rabbit anti-human C3d antiserum (I) was used in the second antibody .step. Finally a peroxydase conjugated anti-rabbit Ig antiserum (j) was added. Two, 2-azino-di(J-ethyl)-benzthiazoline sulphonic acid (ABTS) was used as substrate. Zymosan activated serum was used as a standard, defined to contain I ,000 arbitrary units (AU)/ml. The results were measured by a spectrophotometer (k) at 405 nm and analyzed with the "Immunosoft" (k) program. In the incubation experiments C3 activation was measured in a similar ELISA, using a different antibody (13) due to a change of methods at our laboratory. The mouse monoclonal antibody bH6 reacting with a neoepitope expressed in C3b, iC3b and C3c, was used to coat the plates. A rabbit anti-human C3c antiserum (I) was used in the second antibody step. The 2 C3 activation assays have shown close correlation in heterogenous patient populations (13). The terminal complement complex was quantified in an ELISA as described by Mollnes and coworkers ( 14). The plates were coated with a mouse monoclonal antibody (MCaEll) specific for a neoepitope expressed only in activated C9. A rabbit anti-human C5 antiserum (i) was applied in the next antibody step. The final steps were similar to those of the C3 activation assays. In the dynamic experiments the number of white blood cells and hemoglobin (Hgb) were determined in an electronic counter (m). Concentrations of C3 activation products and TCC were corrected for hemodilution by multiplication with the factor: initial Hgb/sample Hgb. The Friedman test ( 15, 299-308) was used for statistical analyses of time-dependent parameter changes for each brand of tubing. The Kruskai-Wallis test (15. 229237) was applied for intergroup comparisons. Results are presented as median with 95% nonparametric confidence interval in brackets. Results--------------Experiments with intact tubings There was a significant rise in C3 activation products compared to baseline after 15 minutes with the Bypass 65 PVC. Latex, Rehau Silicone, and R.X. Silicone tubings (p<O.OOI), whereas the increase induced by the Tygon tubing did not reach significance (p = 0.12) (figure 1). The maximal increase in C3 activation products from baseline ranged from 37% (20-102) to 75% (22-123) for the various brands of tubing and was 8% (0-22) for conj Amersham, U.K. k Dynatech MR 580, Dynatech Laboratories, Alexandria, Virginia, USA Behring, Hamburg, West Germany m Coulter Electronics, Harkenden, England Volume 21, Number 1, Spring 1989 C3 activation (AU/mil