HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Stephen B. Horton Andrew D. Cochrane Christian P. Brizard Tom R. Karl Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Horton, S. B. Articles by Karl, T. R. Search for Related Content PubMed PubMed Citation Articles by Horton, S. B. Articles by Karl, T. R.

Ann Thorac Surg 1999;68:1751-1755
© 1999 The Society of Thoracic Surgeons

---

Original Articles

IL-6 and IL-8 levels after cardiopulmonary bypass are not affected by surface coating

^a Cardiac Surgical Unit, Royal Children’s Hospital, Melbourne, Australia
^b Intensive Care Unit, Royal Children’s Hospital, Melbourne, Australia
^c Pathology Department, Royal Melbourne Hospital, Melbourne, Australia

Address reprint requests to Dr Karl, Cardiac Surgical Unit, Royal Children’s Hospital, Flemington Rd, Parkville, 3052 Victoria, Australia
e-mail: cardiac{at}cryptic.rch.unimelb.edu.au

	Abstract

Top Abstract Introduction Patients and methods Results Comment References

 
Background. Contact of blood with the surfaces of the cardiopulmonary bypass (CPB) circuit has been implicated as a cause of the inflammatory response. We undertook a prospective randomized trial of 200 pediatric patients, all with a calculated total bypass flow of less than 2.3 L/min (< 0.96 L/m²/min).

Methods. Patients were randomly assigned to 1 of 4 CPB groups: (1) Nonheparin-bonded circuit with no albumin preprime; (2) Nonheparin-bonded circuit with albumin preprime; (3) Heparin-bonded circuit with no albumin preprime; (4) Heparin-bonded circuit with albumin preprime. Measurements of cytokines, (interleukin [IL]-6, IL-8) and blood cell counts were made prebypass and 6 and 24 hours after institution of cardiopulmonary bypass.

Results. Analysis of variance showed no significant difference in any of the clinical or biochemical characteristics of the 4 groups. The interaction between heparin-bonded oxygenators and albumin preprime was not significant. No important differences in IL-6 or IL-8 concentrations were noted after CPB using either heparin or nonheparin-bonded oxygenators with albumin or albumin free preprime using two-way analysis of variance.

Conclusions. Albumin preprime and heparin-bonding do not attenuate the inflammatory response component attributable to the concentration of these markers.

	Introduction

Top Abstract Introduction Patients and methods Results Comment References

 
The systemic inflammatory response after cardiopulmonary bypass (CPB) can be characterized by the production of proinflammatory cytokines, which may lead to complications postoperatively [1, 2, 3]. Cytokines are chemical mediators that participate in immunoendocrine interactions, and play an important role in the pathogenesis of septic shock and chronic illness [4, 5]. Not all cytokines are damaging, with transforming growth factor-ß having cytoprotective properties [6]. Recently, attention has been focused on the role of cytokines as mediators of metabolic, immunological, and endocrine responses to operation. Increased levels of certain cytokines have been demonstrated during and after operation, including cardiac operation with CPB, while the secretion of other cytokines seems to be suppressed [7].

It has been postulated that modulating the blood-foreign surface interaction in the oxygenator and other components of the CPB circuit might attenuate the cytokine response to CPB [8]. In order to assess this, we employed albumin coating (albumin preprime) and/or heparin-bonding of the surfaces of the circuit. Measurements of selected cytokine levels prebypass and postbypass as well as various clinical parameters were compared across 4 CPB groups.

	Patients and methods

Top Abstract Introduction Patients and methods Results Comment References

 
After approval by our institutional human ethics committee, and with informed consent of the parents, 200 consecutive pediatric patients with a calculated individual total bypass flow of less than 2.3 L/min were randomized into 1 of 4 groups: (1) Nonheparin-bonded plate membrane oxygenator (our standard oxygenator) with no albumin preprime; (2) Nonheparin-bonded plate membrane oxygenator with albumin preprime; (3) Heparin-bonded hollow fiber oxygenator with no albumin preprime; and (4) Heparin-bonded hollow fiber oxygenator with albumin preprime.

The maximum rated blood flow for the heparin-bonded hollow fiber oxygenator was 2.3 L/min. Therefore for the purpose of uniformity, all patients having a calculated cardiac output greater than 2.3 L/min were excluded from this study. The median weight for all patients was 6.45 kg (range 1.60 to 26.10 kg), and there were no intergroup differences for weight (p = 0.91).

Albumin prepriming consisted of circulating a calculated amount of crystalloid, Plasmalyte 148 and 5% glucose (Baxter Healthcare Pty Ltd, Toongabbie, NSW, Australia) in a ratio of 5 to 1, containing 40 g/L albumin, for 10 minutes through the CPB circuit. This colloid preprime was then partly removed and replaced with blood (heparinized whole blood less than 24 hours old, or fresh whole blood less than 3 days old) according to our normal protocol for patients requiring a blood prime. The preprime was left in the CPB circuit if the patient’s size and hematocrit were sufficient for a colloid prime. A blood prime was used in 94% of the patients in this study, with one unit of blood used per patient. The amount of preprime left in the circuit was calculated for a final hemoglobin concentration of 9 g/dL.

Blood samples were taken immediately prior to CPB, and 6 and 24 hours thereafter, for measurements of interleukin (IL)-6, IL-8, blood gases, and full blood count (FBC). IL-6 and IL-8 have been shown to peak at approximately 4 hours after termination of bypass [9]. Our mean bypass times for all groups ranged from 103 to 135 minutes, thus 6 hours after bypass institution was chosen as most likely to achieve maximum interleukin levels. Twenty four hours was chosen as the time point most likely to reveal the secondary peak of an expected bimodal distribution [1].

Cytokine levels
Blood samples for cytokine determinations were drawn from the arterial line preoperatively, and 6 and 24 hours after initiation of CPB. Blood samples (5 mL) were immediately placed into sterile polypropylene test tubes and centrifuged at 5000 rpm for 15 minutes at 4°C. The plasma was then transferred to a sterile 5 mL polypropylene test tube and stored at -70°C until bioassay.

IL-6 and IL-8 were measured by assays developed in house using commercially available antibodies (R&D Systems, Minneapolis, MN). Microtiter plates were coated for 48 hours with polyclonal antibodies to either IL-6 or IL-8. After washing the plates, diluted samples and standards were added and incubated for 2 hours at room temperature. The plates were again washed, followed by the addition of monoclonal antibody. The plates were incubated for 2 hours at room temperature. Antimouse immunoglobulin antibody conjugated to biotin, was then added. After another 2 hour incubation, streptavidin-horseradish peroxidase conjugate was added and allowed to incubate for 30 minutes. After washing the plates, tetramethyl benzidine was added and color development was allowed to proceed for 15 minutes, after which phosphoric acid was added to stop the reaction. Light absorbance was then read at a wavelength of 450 nm.

Each assay was calibrated against international reference standards (IL-6 88/514, IL-8 89/520). To ensure assay to assay uniformity, a serum control with established cytokine levels was run in each assay. All assays were optimized to minimize the effect of heterophilic antibodies by adding nonimmune sheep serum to all diluents.

Standards were routinely diluted in a phosphate buffered saline and bovine serum albumin solution, to ensure year to year consistency with standard absorbances. Patient results were calculated from a standard curve plotting standard absorbance versus standard concentrations. Both assays were calibrated against NIBSC (National Institute of Biologic Standard and Controls) reference material. The minimal detectable levels for IL-6 and IL-8 were 18 pg/mL and 15 pg/mL, respectively. These were determined by finding the lowest possible standard able to be distinguished from the blank and then doubling it. The average interassay coefficient of variation (CV) is 5%, and the intraassay CV 12%. All antibodies used are commercially available with certified specificity.

Statistical analysis
We used 2-way analysis of variance for the cytokine measurements (Stata release 6.0, Stata Corporation), with log transformation (due to the highly skewed nature of the data, typical of variables that are necessarily positive). Statistical calculations were then performed, with calculation of the antilog of the result.

The baseline characteristics of the 4 groups were compared by one-way analysis of variance (Prism version 2.01, Graphpad Software).

	Results

Top Abstract Introduction Patients and methods Results Comment References

 
Two hundred patients underwent CPB during the study period in a routine manner. Heparin dose was 3 mg/kg with reversal by protamine (3 mg/kg) after the cessation of CPB. Forty-nine percent had heparin-coated oxygenators, and of this group 49% had an albumin preprime. Fifty-one percent had conventional oxygenators, with 49% having an albumin preprime. There was no difference between the 4 groups in patient height (p = 0.76), weight (p = 0.91), or surface area (p = 0.65) (Table 1). Similarly, CPB time (p = 0.29), cross-clamp time (p = 0.71), circulatory arrest time (p = 0.54), and lowest core temperature during CPB (p = 0.08) (Table 2) showed no significant differences.

	Comment

Top Abstract Introduction Patients and methods Results Comment References

The major sites of cytokine synthesis are the cells of the macrophage and monocyte series. Almost every nucleated cell can produce them, following tissue injury. Acting locally at low concentrations they can be protective or damaging. Optimal function of the defense and repair systems of the body are normally dependent on them.

The proinflammatory cytokines (IL-1 alpha, IL-1 beta, tumor necrosis factor alpha, IL-6, and IL-8) under pathological conditions such as major trauma, sepsis, and shock, are potentially harmful mediators, especially when plasma levels are elevated. IL-6 and IL-8 are readily analyzed using an enzyme-linked immunosorbant assay and were selected as markers of the inflammatory response. IL-6 regulates immune responses and hemopoiesis and is the main cytokine released after cardiac operation. Plasma levels correlate to the degree of surgical trauma and are sensitive markers of tissue damage [10, 11]. By measuring preoperative IL-6 levels routinely, patients at risk may be identified, and higher postoperative levels are predictive of patient complications [12].

IL-1 and tumor necrosis factor (TNF), at levels unable to be detected, can stimulate a variety of cells to produce IL-8. This is not specific to cardiac surgery, but is related to ischemia-reperfusion or anoxia-hyperoxia injury [13–15]. IL-8 is a very potent neutrophil chemotactic and activating factor and has been shown to prime human neutrophils for enhanced superoxide production. Elevated levels have been demonstrated in septic shock and after the intravenous administration of small doses of endotoxin [16, 17]. The level increases in relation to major operation with a response time parallel to that of IL-6. Pulmonary production by alveolar macrophages may be a major source, as high levels of IL-8 in the bronchoalveolar lavage fluid of patients after CPB have been reported [14, 18].

Others have recognized activation of leucocytes with subsequent release of inflammatory mediators during CPB. Gu and colleagues [19] concluded from their study of 30 patients using Duraflo II, that surface heparin coating did not reduce C3a generation during CPB but did reduce the increase in C3a after protamine administration. Also TNF concentration did not increase during the initial period of CPB in either the Duraflo II or the control group. However, TNF concentration increased significantly after protamine administration in the control group and not in the Duraflo II group.

Steinberg and associates [20] measured IL-1, TNF-alpha, IL-6 and IL-8 in 20 patients (6 of whom had heparin bonded circuits). The age of patients was 72 ± 5.3 years for heparin bonded circuits and 66 ± 4.5 years for nonbonded circuits. They found no elevation of IL-1 or TNF-alpha in either patient group, but there was a significant increase in both IL-6 and IL-8 for the nonbonded circuits with return toward baseline 24 hours postbypass. There was no assessment of clinical differences, but due to the skewed nature of their data, comparing the 2 groups with repeated measures of analysis of variance might not have been appropriate. This raises questions regarding their conclusions, and could explain the apparent conflict with our own results. When comparing mean cytokine levels in Steinberg’s data and our own, the IL-6 and IL-8 levels in our study are much lower at similar times, than in either group reported by Steinberg. This may reflect the different populations (pediatric versus adult), differences in bypass technique, differences in the oxygenator used or differences in the method of laboratory analysis.

Recently studies have been undertaken by Ashraf and associates [21] and colleagues [22] and Schreurs examining cytokine response after heparin-bonded CPB. Both studies indicate that a larger patient population is needed to clarify discrepancies. Their study groups consisted of 21 and 19 patients, respectively. The Ashraf study suggests a reduced inflammatory response in terms of IL-6, C5b-9, and elastase levels, which correlates to a reduced postoperative ventilation time. However the investigators found no significant difference for IL-8 and neutrophil counts, with only 1 point (24 hours) being significantly different for IL-6. On examination of the error bar overlap between the groups, it makes the optimistic claim that there is a reduced proinflammatory response (in terms of IL-6).

Schreurs and associates found no significant difference between the groups concerning intraoperative and postoperative blood loss, transfusion requirements, urine production, kidney or liver function, or duration of postoperative ventillatory support. Likewise, no significant differences were found for postoperative gain in body weight or complement activation. SE-selectin at 4 time points and ß-thromboglobulin at 1 time point reached significance with point-by-point analysis, however area under the curve analysis was not significant. Postoperative central body temperature was less elevated in the heparin bonded group with point-by-point analysis, but areas under the curves were similar.

As these authors state, complex biological system studies with large standard deviations in a limited number of patients can make meaningful analysis difficult. We elected to undertake such a large study to help clarify these issues.

Misoph and associates [23], in their study comparing the effect of roller pump, centrifugal pump, uncoated, and heparin-coated surfaces on 73 patients, determined that CPB affects the cellular immune system independent of the type of CPB circuit system.

We measured both cytokine levels and common clinical parameters to ascertain whether a patient benefit could be conferred. We found no important differences between groups, which is at variance with other published studies. However, our study of 200 patients is larger than any previous study. This study used 2 different types of oxygenators. It is possible that the plate membrane device causes less surface activation than the hollow fiber (heparin-bonded) device. The lack of apparent effect of heparin bonding could disguise a poorer performance by the hollow fiber oxygenator. However, our conclusion regarding lack of effect of albumin preprime applies to both types of oxygenator.

By undertaking a large study, we have been unable to show clear differences (either clinically or biologically) in the parameters measured. IL-6 and IL-8 levels after CPB were not influenced by modulation of the blood-foreign surface interface with albumin coating or heparin bonding.

	References

Top Abstract Introduction Patients and methods Results Comment References

 

1. Hennein H.A., Ebba H., Rodriguez J.L., et al. Relationship of the proinflammatory cytokines to myocardial ischemia and dysfunction after uncomplicated coronary revascularization. J Thorac Cardiovasc Surg 1994;108:626-635.[Abstract/Free Full Text]
2. Seghaye M.C., Grabitz R.G., Duchateau J., et al. Inflammatory reaction and capillary leak syndrome related to cardiopulmonary bypass in neonates undergoing cardiac operations. J Thorac Cardiovasc Surg 1996;112:687-697.[Abstract/Free Full Text]
3. Ito H., Hamano K., Gohra H., et al. Relationship between respiratory distress and cytokine response after cardiopulmonary bypass. Surgery Today 1997;27:220-225.[Medline]
4. Glauser M., Zanetti G., Baumgartner J., et al. Septic shock. Lancet 1991;338:732-736.[Medline]
5. Fong Y., Lowry S. Tumor necrosis factor in the pathophysiology of infection and sepsis. Clin Immunol Immunopathol 1990;55:157-170.[Medline]
6. Lefer A., Tsao P., Aoki N., et al. Mediation of cardioprotection by transforming growth factor-beta. Science 1990;249:61-64.[Abstract/Free Full Text]
7. Sablotzki A., Dehne M., Welters I., et al. Alterations of the cytokine network in patients undergoing cardiopulmonary bypass. Perfusion 1997;12:393-403.[Abstract/Free Full Text]
8. Von Segesser L.K. Surface coating of cardiopulmonary bypass circuits. Perfusion 1996;11:241-245.[Free Full Text]
9. Tonneson E., Vibeke C.B., Palle T. The role of cytokines in cardiac surgery. International Journal of Cardiology 1996;53(Suppl):S1-S10.
10. Decker D., Lindemann C., Low A., Bidlinghaier F., Hirner A., von Ruecker A. Changes in the cytokine concentration (IL-6, IL-8, IL-1 RA) and their cellular expression of membrane molecules (CD25, CD30, HLA-DR) after surgical trauma. Zentralblatt fur Chirurgie 1997;122:157-163.[Medline]
11. Jiang J.X., Tian K.L., Chen H.S., Zhu P.F., Wang Z.G. Plasma cytokines and endotoxin levels in patients with severe injury and their relationship with organ damage. Injury 1997;28:509-513.[Medline]
12. Donati A., Battisti D., Recchioni A., et al. Predictive value of interleukin 6 (IL-6), interleukin 8 (IL-8) and gastric intramucosal pH (pH-i) in major abdominal surgery. Inten C Med 1998;24:329-335.
13. Finn A., Naik S., Klein N., et al. Interleukin-8 release and neutrophil degranulation after pediatric cardiopulmonary bypass. J Thorac Cardiovasc Surg 1993;105:234-241.[Abstract]
14. Jorens P., de Jongh R., de Backer W., et al. Interleukin-8 production in patients undergoing cardiopulmonary bypass. Am Rev Respir Dis 1993;148:890-895.[Medline]
15. Metinko A., Kunkel S., Standiford J., et al. Anoxia-hyperoxia induces monocyte derived interleukin-8. J Clin Invest 1992;90:791-798.
16. Khair O.A., Davies R.J., Devalia J.L. Bacterial-induced release of inflammatory mediators by bronchial epithelial cells. Euro Resp J 1996;9:1913-1922.
17. Soto A., Evans T.J., Cohen J. Proinflammatory cytokine production by human peripheral blood mononuclear cells stimulated with cell-free supernatants of Viridans streptococci. Cytokine 1996;8:300-304.[Medline]
18. Jorens P., Van Damme J., de Backer W., et al. Interleukin-8 (IL-8) in the bronchoalveolar lavage fluid from patients with the adult respiratory distress syndrome (ARDS) and patients at risk for ARDS. Cytokine 1992;4:592-597.[Medline]
19. Gu Y.J., van Oeveren W., Akkerman C., Boonstra P.W., Huyzen R.J., Wildevuur C.R.H. Heparin-coated circuits reduce the inflammatory response to cardiopulmonary bypass. Ann Thorac Surg 1993;55:917-922.[Abstract]
20. Steinberg B.M., Grossi E.A., Schwartz D.S., et al. Heparin bonding of bypass circuits reduces cytokine release during cardiopulmonary bypass. Ann Thorac Surg 1995;60:525-529.[Abstract/Free Full Text]
21. Schreurs H.H., Wijers M.J., Gu J., et al. Heparin-coated bypass circuits. Ann Thorac Surg 1998;66:166-171.[Abstract/Free Full Text]
22. Ashraf S., Yi T., Cowan D., et al. Release of proinflammatory cytokines during pediatric cardiopulmonary bypass. Ann Thorac Surg 1997;6:1790-1794.
23. Misoph M., Schwender S., Babin-Ebell J. Response of the cellular immune system to cardiopulmonary bypass is independent of the applied pump type and the use of heparin-coated surfaces. Thorac Cardiovasc Surg 1998;46:222-227.[Medline]

This article has been cited by other articles:

				R. Lorusso, G. De Cicco, P. Totaro, and S. Gelsomino Effects of phosphorylcholine coating on extracorporeal circulation management and postoperative outcome: a double-blind randomized study Interactive CardioVascular and Thoracic Surgery, January 1, 2009; 8(1): 7 - 11. [Abstract] [Full Text] [PDF]

				O. Mangoush, S. Purkayastha, S. Haj-Yahia, J. Kinross, M. Hayward, F. Bartolozzi, A. Darzi, and T. Athanasiou Heparin-bonded circuits versus nonheparin-bonded circuits: an evaluation of their effect on clinical outcomes Eur. J. Cardiothorac. Surg., June 1, 2007; 31(6): 1058 - 1069. [Abstract] [Full Text] [PDF]

				A. M. Draaisma, M. G. Hazekamp, N. Anes, P. H. Schoof, C. E. Hack, A. Sturk, and R. A.E. Dion Phosphorylcholine Coating of Bypass Systems Used for Young Infants Does Not Attenuate the Inflammatory Response Ann. Thorac. Surg., April 1, 2006; 81(4): 1455 - 1459. [Abstract] [Full Text] [PDF]

				A. Boning, J. Scheewe, T. Ivers, C. Friedrich, J. Stieh, S. Freitag, and J. T. Cremer Phosphorylcholine or heparin coating for pediatric extracorporeal circulation causes similar biologic effects in neonates and infants J. Thorac. Cardiovasc. Surg., May 1, 2004; 127(5): 1458 - 1465. [Abstract] [Full Text] [PDF]

				K. Durgut, K. Hosgor, N. Gormus, U. Ozergin, and H. Solak The cerebroprotective effects of pentoxifylline and aprotinin during cardiopulmonary bypass in dogs Perfusion, March 1, 2004; 19(2): 101 - 106. [Abstract] [PDF]

				S Horton, C Thuys, M Bennett, S Augustin, M Rosenberg, and C Brizard Experience with the Jostra Rotaflow and QuadroxD oxygenator for ECMO Perfusion, January 1, 2004; 19(1): 17 - 23. [Abstract] [PDF]

				E. Jensen, S. Andreasson, A. Bengtsson, H. Berggren, R. Ekroth, L. Lindholm, and J. Ouchterlony Influence of two different perfusion systems on inflammatory response in pediatric heart surgery Ann. Thorac. Surg., March 1, 2003; 75(3): 919 - 925. [Abstract] [Full Text] [PDF]

				J. Butler, E. W. Murithi, V. L. Pathi, K. J.D. MacArthur, and G. A. Berg Duroflo II heparin bonding does not attenuate cytokine release or improve pulmonary function Ann. Thorac. Surg., July 1, 2002; 74(1): 139 - 142. [Abstract] [Full Text] [PDF]

				E. R. Stephenson Jr and J. L. Myers Pediatric cardiopulmonary bypass Ann. Thorac. Surg., December 1, 2001; 72(6): 2176 - 2177. [Full Text] [PDF]

				E. A. Grossi, C. Derivaux, and A. LaPietra Heparin coating of bypass systems Ann. Thorac. Surg., July 1, 2000; 70(1): 335 - 335. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Stephen B. Horton Andrew D. Cochrane Christian P. Brizard Tom R. Karl Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Horton, S. B. Articles by Karl, T. R. Search for Related Content PubMed PubMed Citation Articles by Horton, S. B. Articles by Karl, T. R.