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Ann Thorac Surg 2008;85:530-535. doi:10.1016/j.athoracsur.2007.08.050
© 2008 The Society of Thoracic Surgeons
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Original Articles: Cardiovascular

Effect of Pericardial Blood Processing on Postoperative Inflammation and the Complement Pathways

Bertrand Marcheix, MD, MS, Michel Carrier, MD*, Catherine Martel, MS, Mariève Cossette, MD, Michel Pellerin, MD, Denis Bouchard, MD, Louis P. Perrault, MD, PhD

Departments of Cardiovascular Surgery and Biostatistics, Biomedical Laboratory, Montreal Heart Institute and University of Montreal, Montreal, Quebec, Canada

Accepted for publication August 22, 2007.

* Address correspondence to Dr Carrier, Department of Surgery, Montreal Heart Institute, 5000 Belanger Street, Montreal, PQ H1T 1C8, Canada (Email: michel.carrier{at}icm-mhi.org).


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 References
 
Background: The objective of the present study was to determine the effect of processing of pericardial blood with a cell-saving device (CS) and vacuum-assisted cardiopulmonary bypass (VACPB) on reduction of postoperative inflammation.

Methods: One hundred patients who underwent on-pump coronary artery bypass grafting surgery were included in a prospective randomized study. Patients were randomly assigned into four groups of 25 patients, each in a two-by-two factorial design: group A had no CS and no VACPB, group B had VACPB alone, group C had CS alone, and group D had CS and VACPB. The complement factors C4a, C3a, and C5a, and the terminal complex sC5b-9, MBL (mannose-binding lectin), and Bb were measured in plasma preoperatively and at 30 and 240 minutes after termination of CPB.

Results: Mean age, CPB, and aortic cross-clamping times were similar in all groups. At 30 and 240 minutes after CPB, C3a, sC5b-9, and Bb were increased and C5a and MBL levels were decreased compared with preoperative levels in all groups. At 240 minutes, Bb levels were lower in patients with CS (p = 0.0002).

Conclusions: The present study shows that contemporary CPB remains associated with a striking activation of all complement pathways and its terminal component. The use of CS decreases the activation of the complement alternative pathway.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 References
 
The systemic inflammatory response syndrome after exposure to cardiopulmonary bypass (CPB) contributes to the development of postoperative complications such as respiratory failure, renal dysfunction, bleeding disorders, and multiorgan failure [1]. Prevention of these adverse events remains a constant challenge for cardiac surgeons. Several attempts have been made to reduce systemic inflammatory response syndrome such as the use of the serine protease inhibitor aprotinin, of heparin-coated circuits, the suppression of pericardial aspiration, and more recently, the use of miniaturized closed CPB circuits. Little evidence is available concerning the effectiveness of one technique over another. Moreover, the pathophysiologic mechanisms and the impact of these techniques on inflammation have rarely been addressed. The use of cell-saving (CS) devices to process pericardial blood and the addition of vacuum-assisted venous return to the extracorporal circuit (VACPB) to enhance blood return to the circuit and decrease venous pressure constitute recent improvements of modern CPB [2, 3]. Although CS was shown to decrease the use of blood products during cardiac surgery, the impact of CS on the postoperative inflammatory response is controversial and no study is available concerning the effect of VACPB on inflammation.

The objective of this prospective randomized study was to determine whether the use of CS and VACPB decreased the postoperative inflammatory response, with a specific focus on the implication of the complement pathways.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 References
 
Design of the Study
Between December 2003 and July 2005, 100 patients undergoing isolated on-pump coronary artery bypass graft surgery (CABG) were included in a prospective randomized study. All patients with valvular and aortic surgery were excluded. The protocol was reviewed and approved by the Ethics Committee of the Research Center at the Montreal Heart Institute. Written informed consent was obtained from all patients. Patients were randomly assigned by a two-by-two factorial design into four groups of 25 patients each: group A had no VACPB and no CS, group B had VACPB alone, group C had CS alone, and group D had VACPB and CS. Randomization was based on random numbers in a sealed card opened by perfusionists just before surgery.

Cardiopulmonary Bypass Circuit and Use of Aprotinin
The cardiopulmonary bypass circuit consisted of a solid venous reservoir with a venous filter (40-µm filter), a hollow-fiber membrane oxygenator (Sorin Biomedical, Mirandola, Italy), nonpulsatile roller pumps (Stockert, Shiley, Munich, Germany), and an arterial filter of 32 µm (Capiox CX AF01; Terumo, Ann Arbor, MI). A standard Hammersmith full-dose protocol of aprotinin (Trasylol; Bayer, Toronto, Ontario), 2 x 106 kallikrein inhibitor units (KIU) in the pump prime, 2 x 106 KIU loading dose, followed by 0.5 x 106 KIU/h was used in all patients.

Vacuum-Assisted Venous Return and Cell-Saving Device
Patients of groups B and D underwent CPB with VACPB. The VACPB system consisted of a Baxter suction regulator (Baxter Health Corporation, Irvine, California). When used, the negative pressure applied to the solid venous reservoir and right atrial cannula was maintained between –5 and –15 mm Hg during surgery. Patients in groups C and D underwent collection of the suctioned blood from the pericardial cavity to a hard-shell cardiotomy reservoir through a 33-µm filter and processing of pericardial blood through a cell-saving device. The processed red blood cells were reinjected intravenously immediately after weaning from CPB. Patients in groups A and B received retransfusion of suctioned blood during CPB.

Biological Study of Postoperative Inflammatory Response
Blood samples were obtained before initiation of CPB, and 30 and 240 minutes after weaning from CPB. Blood samples were collected in tubes containing ethylenediamine tetra-acetic acid (EDTA) and placed on ice to be rapidly centrifuged at 4°C for 15 minutes at 3,500 rpm. The plasma aliquots were kept at –70°C until the analyses for mannose-binding lectin (MBL) forming the lectin pathway (MBL pathway), C3a from the three activation pathways, classical, alternative, and lectin, C4a more specific for the classical, and lectin pathway, and Bb specific to the alternative pathway. The C5a and the complex sC5b-9 from the terminal activation pathway were also assessed (Fig 1). Commercially available solid phase sandwich enzyme-linked immunosorbent assay (ELISA) tests were used to determine plasma levels of complement proteins Bb and sC5b-9 (BD PharMingen OptEIATM/Quidel, San Diego, California) following the manufacturer’s recommendations. A double-sandwich ELISA for MBL (MBL Oligomer ELISA kit, KIT 029; AntibodyShop, Copenhagen, Denmark) was used. In this capture assay, the antibody combination is selective for normally oligomerized MBL forms; MBL concentrations are thus closely correlated with MBL function when complement is activated [4]. The anaphylatoxins C3a, C4a, and C5a plasma levels were measured with a particle-based immunoassay (BD Biosciences-Immunocytometry Systems, San Jose, CA) [5]; in this test, EDTA plasma is incubated with beads for 2 hours, then washed and incubated for 1 hour with the PE-conjugated detection antibodies; the sandwich complexes are detected by flow cytometry (Coulter EPICS XL Flow Cytometer; Beckman Coulter, Miami, FL).


Figure 1
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  		Fig 1. Diagram of the complement pathways. The black arrows indicate the complement fractions of interest for this study. (MBL = mannose-binding lectin.)

 
Statistical Analysis
Baseline characteristics are presented overall and according to cell-saving device and vacuum-assist groups. Data were presented as mean ± SD for continuous variables, except for not normally distributed data, which are presented as median (minimum-maximum). Frequency and percentage were presented for categorical variables. Patient characteristics were compared between groups using {chi}2 test in case of categorical variables whereas continuous variables were compared using one-way analysis of variance (ANOVA) or Kruskall-Wallis test if distributional assumptions were not met. Three-way repeated ANOVA was used to study the biochemical variables across time and between cell-saving device and vacuum-assist groups. The repeated measures ANOVA was applied to determine if there was interaction between the three main effects. In case of a significant interaction, contrasts were used to compare cell-saving device and vacuum-assist groups at each time. Otherwise, global conclusions were drawn based on the main time, cell-saving device, and vacuum-assist effects of the model. All analyses were done with SAS version 8.2 (SAS Institute, Cary, North Carolina) and conducted at the 0.05 significance level.


   Results
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 Abstract
 Introduction
 Material and Methods
 Results
Comment
 References
 
Patient Characteristics and Operative and Postoperative Data
Patient age, sex ratio, previous history of hypertension, diabetes mellitus, unstable angina, Parsonnet score, and history of myocardial infarction are shown in Table 1. There were no statistically significant differences among the four groups. Cardiopulmonary bypass time and aortic cross-clamping time were similar in the four groups.


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  		Table 1 Patient Characteristics, Operative Data, and Postoperative Course
 
Complement Classical, Common, and Terminal Pathway
The C4a levels increased significantly 30 minutes after CPB compared with preoperative levels (p = 0.0065) and decreased to baseline levels 240 minutes after CPB (p < 0.0001 from 30-minute level; Table 2). The C3a levels increased significantly 30 minutes after CPB compared with preoperative levels (p < 0.0001). The C3a levels decreased at 240 minutes compared with level at 30 minutes (p < 0.0001) but remained significantly higher than preoperative levels (p < 0.0001). The C5a levels decreased 30 minutes after CPB compared with preoperative C5a levels (p < 0.0001) and remained stable as long as 240 minutes after CPB. The sC5b-9 levels increased significantly 30 minutes after CPB compared with preoperative levels (p < 0.0001) and decreased 240 minutes after CPB (p < 0.0001). There was no significant difference in C4a, C3a, C5a, and sC5b-9 levels between groups throughout the study.


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  		Table 2 Kinetics of C4a, C3a, C5a, sC5b-9, MBL, and Bb in EDTA Plasma Preoperatively (0) and 30 and 240 Minutes After Weaning From Cardiopulmonary Bypass in the Four Groups
 
Complement Lectin and Alternative Pathway
Mannose-binding lectin levels decreased 30 and 240 minutes after CPB compared with preoperative level (p = 0.0064 and p = 0.0057; Table 3). There was no difference between groups throughout the study. The Bb levels increased significantly 30 minutes after CPB compared with preoperative levels in all patients (p < 0.0001); Bb levels 240 minutes after CPB were lower in patients in groups C and D compared with patients in groups A and B (p = 0.002). The interaction between the cell-saving device (groups C and D) and time factors was significant (p = 0.0019), suggesting that patients in whom the cell-saving device was used had different Bb levels throughout the study period compared with non-CS groups.


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  		Table 3 The p Values of the Three-Way Repeated ANOVA Study
 

   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
References
 
Cardiopulmonary bypass remains a major factor in the postoperative development of systemic inflammatory response syndrome and its complications after cardiac surgery [1]. The inflammatory response to CPB is initiated by contact between heparinized blood and nonendothelial cell surface and is part of the body’s defense reaction [6–8]. Although far from completely described and understood, this inflammatory response is associated with the development of postoperative complications such as respiratory failure, renal dysfunction, bleeding disorders, and multiorgan failure. One of the principal blood elements involved in this acute defense reaction is the complement plasma protein system. The complement system constitutes a group of more than 30 plasma proteins that interact to produce powerful vasoactive anaphylatoxins, C3a, C4a, and C5a, and the terminal complement complex sC5b-9 [8]. The complement system is known to be activated at three different time points during CPB and cardiac surgery: first, during blood contact with nonendothelial cell surfaces [7–9]; second, after reperfusion of the ischemic arrested heart [8–12]; and third, after protamine administration and formation of the protamin-heparin complex [7–10]. Previous studies have focused on the classical and alternative pathways [7, 13]. Cardiopulmonary bypass and myocardial reperfusion activate the complement through both classical and alternative pathways whereas the heparin-protamine complex activates complement by the classical pathway [7, 9]. Other agonists activating the classical pathway during CPB include endotoxins [13], apoptotic cells, and C-reactive protein [8]. The role of the recently discovered lectin pathway in the postoperative inflammatory response after on-pump CABG has not been studied.

The present study shows that the complement classical and lectin pathways are activated in all groups of patients. The complement alternative pathway is also activated in all groups of patients, but lower levels of Bb were observed after CPB in patients who underwent CABG with the use of a cell-saving device to process pericardial blood during surgery. Processing pericardial blood with a cell-saving device has been shown to protect patients from embolic neurologic injury and to help blood conservation during surgery. Several authors have suggested that reinfusion of pericardial blood is in part responsible for the systemic inflammatory response [14, 15]. Although Aldea and colleagues [14] showed a decrease in the terminal complement sC5b-9 in patients with the elimination of cardiotomy suction, results of the present study showed that processing pericardial blood with cell-saving devices reduces the postoperative Bb increase in plasma levels without affecting the other complement pathways.

The three proximal complement pathways (classical, lectin, alternate) were activated by CPB in all patients. Thirty minutes after weaning from CPB, the C3a, C4a, and Bb levels increased significantly. These proximal pathways lead to the activation of the terminal pathway. Thus, sC5b-9 level was increased 30 minutes after CPB. The formation of the soluble terminal membrane attack complex acted in parallel with the classical pathway activation; after increasing within the first 30 minutes, it decreased significantly from 30 to 240 minutes.

The Bb acted in a different manner by increasing at 30 minutes and remaining elevated 240 minutes after CPB. Complement activation is known to occur in the initial phase of CPB, primarily through the alternative pathway, and the rapid increase in Bb early after initiation of CPB illustrates this alternate pattern of complement activation [16].

Although the classical and alternative pathways are known to be activated with CPB, the role for the mannose-lectin pathway, the most recently discovered complement pathway, has not been well elucidated [7, 13]. Mannose-binding lectin, also called mannam-binding lectine, is a calcium-dependent serum protein that binds to carbohydrates on the surface of a wide range of pathogens or acts directly as an opsonin. Mannose-binding lectin, a member of the collectin family of proteins, can opsonize bacteria by tagging the surface of a pathogen to facilitate recognition and ingestion by phagocytes [17, 18]. It is antibody independent and bypasses C1q and the classical pathway in generating C3a and C5a. In the present study, MBL consumption in parallel with an increase in C3a and C4a levels 30 minutes after weaning from CPB could be related with activation of the lectin pathway during CPB.

We have shown that the use of CS to process pericardial blood reduces activation of the alternative pathway, with lower levels of Bb 240 minutes after weaning from CPB. The use of CS allows the red blood cells to be isolated without bringing platelets and polynuclear cells. With CPB, as discussed previously, complement activation is known to occur primarily through the alternative pathway [13, 19, 20]. Being initiated first, more Bb proteins could be formed within 30 minutes, compared with products from the other proximal pathways. It must be underlined that the Bb increase is easier to detect compared with the other proximal proteins, making the effect of CS also easier to detect. Moreover, as the complement alternative pathway is initiated by "foreign" surface contact partly in the pericardial cavity, CS processing allows retransfusion of washed erythrocytes, and consequently washout of activated complement split products. Thus, the alternative pathway remains activated only by the feedback loop, resulting in a lesser activation of the alternative pathway.

In conclusion, this prospective randomized study confirms that modern CPB techniques remain associated with a strong activation of all complement pathways, including the terminal component. The use of a cell-saving device to process pericardial blood was suggested to reduce the risks of neurologic complications and of exposure to allogeneic blood products [2, 21]. We also demonstrated that when used, a cell-saving device decreased the activation of the complement alternative pathway. This biological study reinforces the interest of systematic use of these adjuncts to classical cardiopulmonary bypass techniques. Further studies remain mandatory to determine the real clinical impact of these biological effects.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 References
 

  1. Levy JH, Tanaka KA. Inflammatory response to cardiopulmonary bypass Ann Thorac Surg 2003;75(Suppl):715-720.
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  8. Walport MJ. Complement N Engl J Med 2001;344:1058-1066.[Free Full Text]
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  12. Chenoweth DE, Cooper SW, Hugli TE, Stewart RW, Blackstone EH, Kirklin JW. Complement activation during cardiopulmonary bypass: evidence for generation of C3a and C5a anaphylatoxins N Engl J Med 1981;304:497-503.[Abstract]
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Ann. Thorac. Surg., November 1, 2008; 86(5): 1723 - 1723.
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