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European Journal of Cardio-thoracic Surgery 11 (1997) 645 – 649 Serum S-100 protein concentration after cardiac surgery: a randomized trial of arterial line filtration David P. Taggart a,*, Kausik Bhattacharya a, Niki Meston a, Susan J. Standing a, Johnathon D.S. Kay a, Ravi Pillai a, Per Johnssson b, Stephen Westaby a a Oxford Heart Centre and Department of Clinical Biochemistry, John Radcliffe Hospital, Oxford OX3 9DU, UK b Department of Cardiothoracic Surgery, Uni6ersity Hospital of Lund, Lund, Sweden Received 24 April 1996; received in revised form 7 August 1996; accepted 15 August 1996 Abstract Introduction: Embolization of gaseous and particulate matter is incriminated in the neuropsychological morbidity of CPB and can be reduced by membrane oxygenators and arterial line filtration. It is not known if the use of arterial line filtration in conjunction with membrane oxygenators might have an additive effect in reducing cerebral injury. Methods: Forty patients undergoing elective coronary artery surgery were prospectively randomized to a 43 mm heparin coated arterial line filter (Cobe Sentry™) or to no filtration (control group). All operations were performed by one surgeon (DPT) using intermittent ischaemia with nonpulsatile CPB, a COBE CML membrane oxygenator and alpha-stat paCO2 management. Flow rates were maintained between 2.0 and 2.4 l − 1 m2 per min with a perfusion pressure of 50 – 80 mmHg and a systemic temperature of 34°C. Cerebral injury was defined by careful neurological examination and serial measurement of the serum concentration of S-100 protein (a highly specific astroglial cell derivative, elevated serum levels of which correlate with proven cerebral injury). Results: There was no difference [mean (S.D.)] in the control and filter groups with respect to age [61(9) vs. 62(9) years], ejection fraction, number of grafts [2.8(0.6) vs. 2.6(0.7)] or CPB times [55(19) vs. 57(18) min]. Preoperatively, no patient had detectable S-100. In the postoperative period 23 of 40 patients (58%) showed elevated S-100 levels. At 1, 5 and 24 h the respective number of patients in the control and filter groups with elevated S-100 was (14 vs. 9), (4 vs. 0), (4 vs. 0)) (P B0.05). No patient had overt cerebral injury. Conclusions: This study suggests that (i) subclinical cerebral injury is common (58% of patients in this study) even after apparently uncomplicated surgery with short CPB times; (ii) serum S-100 protein is a valuable marker for investigating potentially cerebral protective innovations during CPB; and (iii) arterial line filtration significantly reduces but does not eliminate cerebral injury. © 1997 Elsevier Science B.V. Keywords: S-100; Cardiopulmonary bypass; Filter; Arterial line filtration; Cerebral injury 1. Introduction Cardiopulmonary bypass (CPB) generates gas bubbles, biological aggregates, atheromatous debris and inorganic matter [12] and microembolization of these substances is incriminated as a major aetiological factor in the neuropsychological morbidity of CPB [6,20]. * Corresponding author. Tel.: + 44 865 221121; fax: + 44 865 220244. 1010-7940/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 1 0 1 0 - 7 9 4 0 ( 9 6 ) 0 1 1 0 3 - 7 The potential for arterial line filtration to reduce neuropsychological morbidity after CPB has been controversial and is dependent not only on the presence or absence of a filter but on the type of oxygenator employed. The efficacy of filtration in removing debris from the extracorporeal perfusate was established more than 20 years ago [4] but concerns were raised that small pore filters might contribute to the generation of embolic aggregates [7] and air trapping [23]. In a trial of a 20 mm filter used in conjunction with a bubble oxygenator, Aris and colleagues could find no differ- 646 D.P. Taggart et al. / European Journal of Cardio-thoracic Surgery 11 (1997) 645–649 ence in neurological or neuropsychological morbidity between the filtered and control groups [3]. Demonstration of cerebral microemboli using retinal fluorescein angiography [6] and middle cerebral artery Doppler ultrasonography [15] has, however, swung the surgical pendulum in favour of membrane oxygenators and arterial line filtration. Using transcranial Doppler ultrasonography Padayachee and colleagues reported a reduction in the cerebral embolic load with membrane rather than bubble oxygenators and suggested that much of the embolic load might be due to gas bubbles [10,11]. Subsequently a number of studies have confirmed that membrane oxygenators reduce the neuropsychological morbidity of CPB [15,17,18]. Recently Pugsley and colleagues reported a reduction in the incidence of microemboli, detected by transcranial Doppler ultrasonography, when a 40 mm arterial line filter was used with a bubble oxygenator [16]. More importantly, this strategy resulted in a reduction in both neurologic and neuropsychological deficits following CPB [16]. To our knowledge there has been no trial of arterial line filtration used in conjunction with membrane oxygenation on cerebral injury following CPB. Forty patients undergoing coronary artery bypass grafting (CABG) with membrane oxygenators were randomized to arterial line filtration or to no filtration. Cerebral injury was defined by neurological examination and serial measurement of the serum concentration of S-100 protein a specific astroglial cell protein [9] whose elevated serum concentration correlates with proven cerebral injury [13,14] and which has been reported to be elevated after CPB [1,19,21,22]. 2. Methods 2.1. Patients Forty patients undergoing elective first time CABG with membrane oxygenators were randomized to arterial line filtration or to no filtration. In the filtered group a 43 mm heparin coated filter (Cobe Sentry™ Arterial Filter with PrimeGard™) was placed in the arterial line of the CPB circuit. Patients with a previous history of cerebrovascular accident, transient ischaemic attacks or with marked renal impairment were excluded. 2.2. Surgery All operations were performed by one surgeon (DPT) using intermittent ischaemia and induced fibrillation. CPB was performed with a roller pump and nonpulsatile flow between 2.0 and 2.4 l − 1 m2 per min at a temperature of 34°C. A COBE CML membrane oxy- genator and alpha-stat paCO2 management were employed and arterial pressure maintained at 50–80 mmHg. 2.3. Blood sampling Blood samples for S-100 protein were collected before anaesthesia, at skin closure and at 5 and 24 h postoperatively. Samples were centrifuged to separate the serum which was frozen at − 20°C for batch analysis. 2.4. S-100 assay Serum S-100 levels were measured by a monoclonal two site immunoradiometric assay (Sangtec 100, Sangtec Medical AB, Bromma, Sweden) which uses three monoclonal antibodies SMST 12, SMSK 25, and SMSK 28 to detect the S100bb and ab dimers which are specific for astroglial cells. After dilution in a buffer with bovine serum albumin the serum samples were incubated for 1 h with a plastic bead coated with monoclonal anti-S100 antibodies which binds the S100. After washing to remove remove any unbound material the beads were incubated with I125 labelled anti-S-100 antibody which binds to the S-100 bound by the bead antibody. Following a further 2 h incubation period and subsequent washing the amount of radioactive label bound to immobilized S-100 was measured by gamma counter. Calibration of the assay involved rehydrating six lyophilized standards of S-100 concentration with diluent as a zero standard to produce a standard curve. High and low control samples were provided in the kit. Samples were analyzed in duplicate to reject those with more than 10% variation. Any duplicate elevation in S-100 above 0.1 mg/l was considered abnormal. 2.5. Postoperati6e examination All neurologic examinations were performed by one observor (KB) blinded as to whether the patient received arterial line filtration or not. 2.6. Statistical analysis Results were analysed using SPSS. Clinical data is presented as means and standard deviations. Biochemical results are presented as medians and inter-quartile ranges and were compared between groups using a Mann-Whitney test. The numbers of patients in each group with elevated S-100 levels ( \ 0.1 mg/l) were compared using a one tail Fisher Exact Test with the probability that arterial line filtration reduces S-100 levels. D.P. Taggart et al. / European Journal of Cardio-thoracic Surgery 11 (1997) 645–649 Table 1 Clinical data in the control and filter groups 4. Discussion Non-filter Filter P Value Number Age (years) Number of grafts CPB time (min) 20 61 (9) 2.8 (0.6) 55 (19) 20 62 (9) 2.6 (0.7) 57 (18) NS NS NS Elevated S100b at: Skin closure 5h 24 h Number of deaths Obvious cerebral Injury 14 4 4 0 0 9 0 0 0 0 647 0.05 0.05 NS NS 3. Results Patient data is presented in Table 1. The groups were very similar with regard to age, ventricular function, ischaemic times, number of grafts and CPB times. Serial changes in S-100 are presented in Fig. 1. S-100 was not detectable in any patient prior to operation but was significantly elevated in both groups (P B0.001) at skin closure [median and interquartile range for the non filter group was 0.3 (0 – 0.55) mg/l compared to 0 (0– 0.39) mg/l in the filter group]. There was no elevation in S-100 level in any patient in the filter group at 5 and 24 h, in contrast to persistently elevated levels in four patients in the non-filter group at the same time points. At skin closure, 5 and 24 h the respective number of patients in the control and filter groups with elevated S-100 was (14 vs. 9), (4 vs. 0), (4 vs. 0)) (P B 0.05). In the non-filter group 6 patients had an S-100 level greater than 0.5 mg/l at skin closure compared with 3 in the filter group but no patient had overt evidence of cerebral injury on clinical examination. Fig. 1. S-100 levels in the control and filter groups before operation, at skin closure and postoperatively at 5 and 24 h. Box plot and whiskers indicate median, inter-quartile range and range. Black dots represent out-liers which are values at least three times greater than the respective inter-quartile range. Morbidity from cerebral injury, both overt and subclinical, continues to limit the outcome after cardiac surgery. A number of studies, reviewed by Smith [20], have reported that 50–60% of patients have neuropsychological deficits 1 week after cardiac surgery and that these abnormalities are still demonstrable in approximately one third of patients 6 months later. Whilst the aetiology of neurological impairment is complex and probably multifactorial, there is considerable circumstantial evidence to implicate microembolization of gaseous and particulate matter [12]. Furthermore, current evidence suggests that methods to reduce the microembolic load generated by CPB may result in improved cerebral outcomes. Such claims have been made independently for arterial line filters [15] and membrane oxygenators [5,16–18] but not in combination. The need for a serum marker of cerebral injury following cardiac surgery was highlighted more than a decade ago when Aberg and colleagues reported a correlation between elevated adenylate kinase levels in the cerebrospinal fluid (CSF) and impairment of intellectual function in patients following CPB [2]. S-100 is a dimeric protein consisting of two subunits (a and b) with a molecular weight of around 21 kDa and a half life in serum of approximately 2 h (9). S-100ao (aa) is found predominantly in striated muscles, heart and kidney while S-100a (ab) is found mainly in astroglial cells and S-100b (bb) is present in high concentrations in astroglial and Schwann cells. The assay used in our study measures both the S-100a (ab) and S-100b (bb) subunits, which are not normally detectable in the circulation. Elevated S-100 protein levels have been reported in the CSF and serum of patients with proven cerebral injury including subarachnoid haemorrhage and stroke [13,14]. Aberg’s group, comparing patients undergoing cardiac surgery with control patients and a mock perfusion circuit with donated blood, concluded that elevated S-100 levels were not due to anaesthesia, surgery or blood trauma but implied cerebral injury [1]. This group also reported that the maximum increase in S-100 was reached at the termination of CPB with return to normal levels shortly after [1]. This time course of acute elevation in S-100 has also been confirmed by Westaby and colleagues [22] and may explain Sellman and colleagues’ failure to detect elevated S-100 levels in CSF 24 h after surgery [19]. Westaby and colleagues confirmed the elevation in serum levels of S-100 after CPB but not in control patients and demonstrated a highly significant correlation with the duration of perfusion [22]. Elevated serum levels of S-100 have also been demonstrated to correlate with the duration of total circulatory arrest despite the use of deep hypothermia and retrograde cerebral perfusion during repair of the aortic arch [21]. 648 D.P. Taggart et al. / European Journal of Cardio-thoracic Surgery 11 (1997) 645–649 In total 23 of 40 patients (58%) in our study showed an elevated S-100 level in the postoperative period. This is similar to the percentage of patients with demonstrable deficits on neuropsychological testing within 1 week of CPB [20]. Our randomized trial showed a benefit of arterial line filtration in terms of S-100 release and by implication cerebral protection, when a heparin-coated arterial line filter was used in conjunction with a membrane oxygenator. In the filter group fewer patients demonstrated elevated S100 levels and both the magnitude and persistence of the increase was less in the filter group. While previous studies have demonstrated improved cerebral outcome with membrane oxygenators compared to bubble oxygenators and arterial line filtration compared to no filtration we are not aware of any other study which has demonstrated a benefit of arterial line filtration in conjunction with a membrane oxygenator. The precise relationship of elevated S-100 levels to subtle abnormalities in intellectual and neuropsychological function has not been clearly defined. In our study because S-100 was not detectable in any patient prior to surgery we considered any elevation of S-100 above 0.1 mg/l, in duplicate samples, abnormal. Furthermore, with reference to the assay the gradient of counts per min against S-100 concentration is very similar below 0.5 mg/l. In our study six patients in the non-filter group and three in the filter group had S-100 levels greater than 0.5 mg/l at skin closure but without overt evidence of cerebral injury. This underlines the need to correlate elevated S100 levels with abnormalities demonstrable on neuropsychological testing and to define an appropriate ‘cut-off’ value for pathologically elevated S-100 levels. It is interesting to speculate on the mechanisms of S-100 release into the systemic circulation and whether this reflects eventual drainage of CSF into the venous circulation or alteration in the integrity of the blood brain barrier. There is little direct evidence of the effects of CPB on the function of the blood brain barrier in man although one animal model failed to demonstrate any disruption of the integrity of the blood brain barrier after 2 h of moderately hypothermic CPB [8]. In summary, our study confirms that S-100 appears to be a valuable marker for the assessment and investigation of subtle cerebral injury following CPB. Our study suggests that subclinical cerebral injury is common (58% of patients in this study) even after apparently uncomplicated surgery with a short period of CPB and that arterial line filtration significantly reduces but does not eliminate this cerebral injury. Acknowledgements We wish to thank Dr Cortina-Borja PhD, Statistician, University of Oxford for expert statistical advice, Anaesthetic and Nursing Staff for assistance in collection of blood samples, Cobe Cardiovascular Inc. for providing the arterial line filters, Cambridge Life Sciences for technical support and Oxford Health Authority for financial support. References [1] Aberg T. Signs of brain cell injury during open heart operations: past and present. Ann Thorac Surg. 1995;59:1312–1315. [2] Aberg T, Ronquist G, Tyden H, Ahlund P, Bergstrom K. Release of adenylate kinase into cerebrospinal fluid during open heart surgery and its relation to postoperative intellectual function. Lancet 1982;1:1139 – 1142. 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