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Eur J Cardiothorac Surg 2004;26:294-300
© 2004 Elsevier Science NL
a Department of Cardiac Surgery, The General Hospital, Southampton, UK
b Department of Cardiac Anaesthesia, The General Hospital, Southampton, UK
c Department of Biomolecular Sciences, University of Portsmouth, Portsmouth, UK
Received 25 December 2003; received in revised form 22 March 2004; accepted 5 April 2004.
* Corresponding author. Address: Department of Cardiac Surgery, Glenfield General Hospital, Groby Rd, Leicester LE3 9QP, UK. Tel.: +44-116-287-1471; fax: +44-116-232-1282
e-mail: alexiou486{at}aol.com
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Abstract |
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 Top Abstract 1. Introduction2. Material and methods3. Results4. DiscussionAppendix A. Conference...References |
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Key Words: Coronary artery bypass grafting • Leucocyte activation and depletion • Lung function
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1. Introduction |
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TopAbstract1. Introduction2. Material and methods3. Results4. DiscussionAppendix A. Conference...References |
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Pulmonary dysfunction is one of the most frequent problems encountered after coronary artery bypass grafting (CABG) surgery [3]. It was believed that the inflammatory response to CPB was the main cause, but it is now recognized that the general anesthesia, surgical injury, co-existing respiratory disease and peri-operative pain are also responsible for this condition [4].
Calculation of alveolar-arterial oxygenation index (AaOI) is an established method for the assessment of peri-operative changes in lung function. More recently, the rate of release of exhaled nitric oxide (NO), a proven marker of pulmonary injury in chronic inflammatory lung conditions (e.g. asthma, bronchiectasis), has been proposed as a sensitive indicator of pulmonary inflammation that occurs during CPB [5].
To address CPB-related organ dysfunction, several anti-inflammatory strategies, including leucocyte depletion, have been used with variable efficacy [6–9].
The ability of the leucocyte-depleting filters to consistently reduce the systemic total white cell counts (WCC) has been questioned, but it has been claimed that these filters may preferentially remove the activated forms of leucocytes [10]. In previous studies [11–13], the impact of leucodepletion on leucocyte activation has been evaluated through measurements of activation markers in plasma. Whilst such markers may provide reliable information on leucocyte activation status, it is likely that the true measure of leucocyte depletion filters should be their ability to remove leucocytes, in particular, activated leucocytes, from the circulation.
The aim of this study was to examine the effect of arterial line leucodepletion on the numbers of activated circulating leucocytes, the pulmonary inflammation and the oxygenation index in low-risk CABG surgery.
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2. Material and methods |
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TopAbstract1. Introduction2. Material and methods3. Results4. DiscussionAppendix A. Conference...References |
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Patients undergoing primary, elective CABG operations were recruited. The following were exclusion criteria: age greater than 70 years, platelet count <75x109/l, WCC <5x109/l, left ventricular ejection fraction <50%, history of lung disease, renal impairment or stroke and treatment with steroid or other anti-inflammatory medications.
Fifty patients were divided in two equal groups using a computerised randomisation system. In the first group (n=25), a leucocyte-depleting filter (LG6, Pall, Portsmouth, UK) was attached onto the arterial line of the CPB circuit. In the control group (n=25), a conventional non-leucocyte depleting filter (D754, Sorin Biomedica, Mirandola, Italy) was used. The management of anaesthesia and CPB, and the conduct of operations were otherwise identical in both groups.
The two groups were compared with regard to the following main end-points: (a) the total and activated WCC at sequential points before, during and after CPB; (b) the rate of alveolar production of exhaled nitric oxide (NO) before and after CPB, and (c) the alveolar-arterial oxygenation index (AaOI) before CPB and up to 18 h post-CPB.
In addition, intra-operative data (aortic cross-clamp time, bypass time, number of distal anastomoses) and other clinical outcome measures (ventilation time, need for inotropic support, occurrence of atrial fibrillation, chest infection, intensive care unit stay and hospital stay) were recorded.
2.1. Management of anaesthesia and conduct of the operations
The patients were pre-medicated with 10 mg of Morphine and 2 mg of Lorazepam. Anaesthesia was induced with Midazolam, Fentanyl and Pancuronium and maintained using intermittent positive pressure ventilation with oxygen-enriched air and isofluorane. During CPB, a Propofol infusion was used to maintain anaesthesia. The CPB circuit consisted of a Dideco D703 (Compactflo) microporous hollow fibre membrane oxygenator (containing a heat exchanger) with integral cardiotomy reservoir (Sorin Biomedica, Mirandola, Italy). The circuit was primed with 2 l lactated Ringer solution that contained 5000 units of heparin. Prior to the establishment of CPB, 3 mg/kg body weight of heparin were administered and supplemented as required to maintain an activated clotting time of 480 s. Continuous non-pulsatile blood-flow was delivered to the patient using a multi-flow roller pump (Stockert SIII, Munich, Germany) at an indexed flow rate of 2.4 l/m2 per min.
After aortic clamping, electromechanical diastolic arrest was induced with the delivery of cold (4 °C) blood cardioplegic solution. Distal anastomoses were completed during a single period of aortic clamping. Proximal anastomoses were performed on a beating heart using an aortic partial occluding clamp. CPB was terminated after the patient was re-warmed having a nasopharyngeal temperature of 37 °C.
2.2. Management in the intensive care unit
After the operation, the patients were kept ventilated until standard extubation criteria were met. The ventilation protocol comprised of 10 breaths/min, tidal volume of 10 ml/kg of body weight, fraction of inspired oxygen of 60%, pressure support of 20 cmH2O, positive end expiratory pressure of 5 cmH2O and inspiratory to expiratory ratio of 1:2. Hydration was achieved with the intravenous administration of Dextrose 5% solution infused at 1 ml/kg/h. Blood, Gelofusine or Human Albumin Solution was given to maintain adequate filling and systemic perfusion pressures and plasma heamoglobin levels above 8.5 g/dl.
2.3. Blood sampling
Blood samples (10 ml) were taken before CPB, 5 min on CPB, 30 min on CPB, 5 min after aortic clamp (AC) removal and 6 h after CPB. The samples before and after CPB were drawn from the radial arterial line and during CPB from the arterial line of the CPB circuit.
2.4. Calculation of total and activated white cell counts
Total WCC were determined manually using the following method. Blood samples (100 µl) were diluted by the addition of 900 µl of 0.2 µm filtered Leucoplate solution (Laboratoires Sobioda SA, Cedex, France). After thorough mixing, the suspension was allowed to stand for 10 min at room temperature until lysis of the red cells occurred. Then, 100 µl of this sample was loaded into a Nageotte counting chamber. The chamber was left to rest in a moistened Petri dish for 15 min to allow the leucocytes to settle. Leucocyte counts were then obtained within the next 30 min using light microscopy at a magnification of x25 (Fig. 1)
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Haemoglobin levels were also determined in order to correct for the effect of haemodilution using a HemoCue photometer that measures directly ß-haemoglobin concentrations.
2.5. Measurement of the alveolar production of exhaled NO
Exhaled NO was measured as parts per billion per second (ppb/sec) using a real-time chemiluminesence analyser (Logan Research, Northampton, UK). The exhaled gas samples were drawn at a sampling rate of 250 ml/min through a Teflon sampling tube sited 1-cm from the tip of the endotracheal tube after stopping the ventilator for 30 s. This maneuver allowed a prolonged exhalation to occur. Three measurements of exhaled NO were taken at 2 min intervals 15 min after median sternotomy (before CPB), and another set of three measurements was taken 30 min after the termination of CPB. The change in the rate or release of exhaled NO during a sustained exhalation was calculated using the formula: 
NO=((CF–CS)/tF–S), where NO is the change in NO (ppb/s), CF is the final concentration of NO (ppb), CS is the start concentration of NO (ppb), tF–S is the sampling time duration (s). An average value for the three measurements of production of exhaled NO (ppb/s) was calculated for each patient both before CPB and after CPB.
2.6. Determination of the alveolar-arterial oxygenation index
To calculate the AaOI, the fraction of inspired oxygen was recorded and a sample of arterial blood was taken from the radial arterial line 30 min before CPB, immediately after CPB and then at 1, 2, 4, 8 and 18 h post-operatively. AaOI for each of the abovementioned time points was determined using the formula: AaOI=((760–47)FiO2–(PaCO2x7.6)1.25))–(PaO2x7.6)/(PaO2x7.6), where FiO2 is the inspired oxygen concentration, PaCO2 is the partial pressure of arterial CO2 (kpa), PaO2 is the partial pressure of arterial O2 (kpa).
2.7. Statistical analysis
Continuous variables were expressed as mean values±standard deviation and the proportions as percentages. The differences between the groups for the categorical variables were compared with 
2 or Fisher's exact test. Continuous variables with normally distributed data were compared with student t-test. If the data were skewed, the Kruskal–Wallis test was used. The overall effect of leucodepletion on WCC and AaOI was assessed by comparing the sequentially obtained data in the two groups with a two-way analysis of variance (ANOVA) with replicates. A P value <0.05 was considered statistically significant. Statistical analysis was performed on Excel 2000 (Microsoft Corp. USA) and SPSS (version 11.5, Chicago, Illinois, USA) software.
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3. Results |
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TopAbstract1. Introduction2. Material and methods3. Results4. DiscussionAppendix A. Conference...References |
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3.5. Alveolar-arterial oxygenation index
The AaOI for the two study groups obtained at different intervals are summarised in Fig. 4
. The two groups had similar AaOI before the initiation of CPB. The AaOI increased following CPB in both groups and remained elevated compared to baseline pre CPB values for up to 18 h post-operatively. However, the leucodepleted group exhibited consistently lower AaOI than the control group overtime (P=0.001).
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4. Discussion |
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TopAbstract1. Introduction2. Material and methods3. Results4. DiscussionAppendix A. Conference...References |
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4.1. Total white cell counts
Compared to control group, the arterial line LG6 filter significantly lowered the total WCC at each sampling point during CPB. Nevertheless, the observed increase in the total WCC in both groups at 5 min after the release of the AC sampling point (when leucocytes that were sequestrated in the pulmonary vasculature re-enter the systemic circulation) indicates that the leucodepleting efficiency of the LG6 filter may be reduced when it is exposed to an increasing load of leucocytes. This would support proposals for selective use of leucodepletion during the period of reperfusion with a view to enhance its potential clinical benefit [14].
These results are in keeping with previous clinical studies reporting a significant reduction of total WCC with the arterial line LG6 filter during CPB [13], but are in contrast to others that described only non-significant changes in the WCC [15], where this was attributed to the rapid replacement of the depleted leucocytes by leucocytes released from bone marrow ‘stores’. Other authors have shown that although the LG6 filter failed to reduce the total WCC during CPB, it attenuated the post-operative leucocytosis [16]. Leucocytosis after cardiopulmonary bypass was, however, present in this and other clinical studies on leucodepletion [13].
4.2. Activated white cell counts
In a study [10], the LG6 failed to reduce the systemic WCC but it appeared to selectively remove activated neutrophils, preventing their sequestration into lungs and other organs. Because of the proven role activated leucocytes have in the end-organ injury sustained by the patients undergoing cardiac surgery with CPB, the hypothesis of the preferential removal of activated leucocytes by the LG6 filter generated considerable interest. There is now experimental and clinical evidence suggesting that preferential removal of activated leucocytes is likely to occur. In an in vitro simulated extracorporeal circuit, the expression of surface adhesion molecules were reduced by around 20% after 60 min of leucodepletion [11]. Immunological analysis of the cells trapped within the filter media that have been used in cardiac surgical procedures, showed that the greater part of the trapped granulocytes were activated granulocytes [17]. In an in vitro simulated extracorporeal circuit run with whole human blood, investigators recorded the removal of total and activated leucocytes over time until further leucocyte removal ceased due, apparently, to a filter saturation effect. They then added Phorbol-Myristate-Acetate, a powerful neutrophil activator, to the circuit, which resulted in a significant increase of activated leucocytes and subsequent complete recovery of leucocyte removal at a rate comparable to that recorded at the beginning of the study [5]. Other workers studied the effect of the metabolic inhibitor sodium azide on the leucodepletion process and observed a reduction in the rate of granulocyte removal by the filter media [18].
In the clinical field, one study reported reduced expression of the CD18 surface adhesion molecule after use of arterial line leucocyte filtration in patients undergoing heart valve operations [12]. A more recent report described a significant reduction in the expression of the adhesion molecules CD11b and L-selectin with continuous arterial line leucocyte depletion in CABG surgery [13]. In the present study, the numbers of activated leucocytes were significantly reduced with arterial line leucodepletion throughout CPB. This is in keeping with the previous reports [12,13] and attests to the ability of the LG6 to remove activated forms of leucocytes during clinical CPB. Six hours after CPB, though, the activated WCC increased in the leucodepleted patients, approximating the values of the controls. It is thus clear that the use of leucodepletion during CPB has no effect on the post-operative total and activated WCC.
4.3. Exhaled nitric oxide
The rate of alveolar production of exhaled NO has increased following CPB in both groups, but this increase was significantly attenuated in the leucodepleted patients.
Exhaled NO increases in chronic inflammatory lung conditions such as asthma in proportion with the severity of the airway inflammation [19], and has been used successfully to monitor the success of anti-asthmatic therapy [20]. Because cardiac surgery induces severe pulmonary inflammation, it would be reasonable to anticipate a rise of the exhaled NO post-operatively.
The most convincing clinical evidence linking systemic inflammation with the levels of exhaled NO has been provided by a prospective randomized study with patients undergoing CABG surgery under CPB [21]. The study recorded a concurrent significant rise of the pro-inflammatory cytokines IL-6 and TNF-
and the exhaled NO, both of which were significantly reduced after administration of systemic steroids [21]. A more recent controlled trial showed a significant increase in the levels of exhaled NO after CPB, which was attenuated in the patients subjected to arterial line leucodepletion with the LG6 filter [5]. The present study is in keeping with the previous reports [5,21]. In addition, it shows that there may exist a relation between the numbers of circulating activated leucocytes, the degree of pulmonary inflammation and the levels of exhaled NO. Other investigators, though, found no increase in the amount of exhaled NO after CABG surgery [22].
4.4. Pulmonary dysfunction
It is well known that activated leucocytes play a crucial role in the development of the post-cardiac surgery pulmonary dysfunction [1–4,6]. The AaOI, an established marker of pulmonary dysfunction, is invariably elevated after cardiac surgery.
In this study, the AaOI increased more than two-fold and remained higher than the baseline values for up to 18 h in both groups. Evaluation of the overall effect of leucodepletion confirmed significantly improved AaOI in the leucodepleted group. Application of arterial line leucodepletion has been found to improve pulmonary function in some [7], but not all [9,23] clinical trials. The novel contribution of this study is that it demonstrates the presence of a cause and effect relationship between the circulating activated leucocytes, the pulmonary inflammation, and the post-operative oxygenation index. Removal of activated leucocytes with an arterial line LG6 filter appears to limit lung inflammation and to ameliorate pulmonary dysfunction.
Although the study is lacking the statistical power to evaluate the effect of leucodepletion on the main clinical outcome end-points, it should be noted that the duration of ventilation, the need for inotropic support, the frequency of occurrence of cardiac and respiratory complications and the time spent in the intensive care unit and the hospital were not improved after leucodepletion. This shows that the low-risk patients with no severe respiratory or other organ co-morbidity are able to compensate for the detrimental effects of cardiac surgery and recover well after their operations.
Nevertheless, improved lung function and reduced mechanical ventilation requirements have been reported after use of leucodepletion in patients having abnormal preoperative pulmonary function, those that were connected to CPB circuit for more than 90 min and after aortic arch surgery under hypothermic circulatory arrest [5,24,25]. These observations imply that the higher risk patients are more likely to derive measurable clinical benefits from the use of continuous arterial line leucodepletion. Thus, future research on the clinical effects of this anti-inflammatory intervention ought to be directed towards such patient groups.
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Acknowledgments |
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Footnotes |
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Appendix A. Conference discussion |
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TopAbstract1. Introduction2. Material and methods3. Results4. DiscussionAppendix A. Conference...References |
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Mr. Alexiou: We are aware of your work showing impaired lung function for several days after coronary bypass surgery with or without cardiopulmonary bypass. This study was limited to the first 18 postoperative hours. We felt that if we had followed up these patients longer it might have been difficult to control for other parameters that influence gas exchange in the lungs such as pain relief, atelectasis, fluid balance etc. We think it is easier to control these variables the first day after the operation.
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References |
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TopAbstract1. Introduction2. Material and methods3. Results4. DiscussionAppendix A. Conference...References |
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This article has been cited by other articles:
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  O. Warren, C. Alexiou, R. Massey, D. Leff, S. Purkayastha, J. Kinross, A. Darzi, and T. Athanasiou The effects of various leukocyte filtration strategies in cardiac surgery Eur. J. Cardiothorac. Surg., April 1, 2007; 31(4): 665 - 676. [Abstract] [Full Text] [PDF]
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 S. C Clark Lung Injury after Cardiopulmonary Bypass Perfusion, July 1, 2006; 21(4): 225 - 228. [Abstract] [PDF]
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