S100 Blood Concentrations in Children Subjected to Cardiopulmonary By-Pass

¹ Dept. of Child Health and Neonatal Medicine, Giannina Gaslini Children's Hospital, 16147 Genoa, Italy;
² Institute of Histology Catholic University, 00168 Rome, Italy;
³ Institute of Anatomy, University of Bari, 70124 Bari, Italy;
⁴ Heart Surgery Service, Quisisana Hospital, 00197 Rome, Italy;
^a author for correspondence: Institute of Histology, Catholic University, Largo F. Vito, 1, 00168 Rome, Italy

S100 is a member of a family of calcium-binding proteins present primarily in nervous tissue, where it is mainly concentrated in glial cells (1)(2). Although the role of this protein in brain function and disease has not yet been conclusively elucidated, it has been ascertained that the appearance of this protein constituent of neural cells in biological fluids is a reliable indicator of active cell damage in the nervous system in different pathological conditions (3)(4)(5)(6).

Open heart surgery is known to be associated with brain cell injury. It was diagnosed previously using rather crude psychometric tests and clinical observations in seemingly healthy patients (7), and measurements of S100 in the blood have recently been successfully used to monitor cerebral damage after cardiac surgery (8)(9)(10).

The present study is aimed at investigating the potential use of measurements of S100 in the blood to monitor possible cerebral distress during extracorporeal circulation in children (0–9 years), when hemodynamic adaptive phenomena in the brain show peculiarities linked to the age of the patient (11). The data presented indicate significantly higher blood concentrations of this nervous tissue constituent during cardiopulmonary by-pass (CPB), together with a direct relationship with the duration of CPB.

Blood samples were taken from 13 patients (6 males and 7 females), ages from <1 year to 9 years (two patients <1 year of age, one 1 year of age, six 3 years of age, one 4 years of age, one 5 years of age, and two 9 years of age), with no preexisting neurological disorders or other co-morbidities, who were undergoing cardiac surgery with CPB for correction of congenital heart disease without emergency procedure. The samples were taken at five predetermined times before, during, and after surgery (time 0, before surgery; time 1, during surgery before CPB; time 2, at the end of CPB; time 3, at the end of surgery; time 4, 12 h after surgery) and measured for S100. Clinical parameters (peripheral temperature, nasopharyngeal temperature, pump flow rate, mean blood pressure, and central and peripheral blood pH) were recorded at all sample times to monitor the general pattern of surgery. Eleven patients had satisfactory postsurgery follow-up, but two patients, whose preoperative general condition was not appreciably different from the others, died as a result of heart failure 36 and 72 h after surgery. The study protocol was approved by the Ethics Committee of Quisisana Hospital, Rome.

Heparin-treated blood samples were immediately centrifuged at 900g for 10 min, and the supernatants were stored at -70 °C before measurement. The S100 protein concentrations were measured in all samples, using a commercially available two-site IRMA (Sangtec 100, AD Sangtec Medical).

Each measurement was performed in duplicate. Either 100 µL of S100 in known concentrations (ranging between 0.5 and 60 µg/L) or 100 µL of patient sample were added to each tube. One hundred microliters of Sangtec 100 IRMA diluent and one plastic bead coated with monoclonal anti-S100 antibody were then added to each tube. The tubes were incubated for 1 h at room temperature (18–20 °C) on a shaker. Each bead was then washed once with 2 mL of demineralized water, and 200 µL of tracer (I-labeled monoclonal anti-S100 antibody) was added to each tube. To estimate total counts, 200 µL of tracer was placed in a tube, and the radioactivity was then counted without additional processing. The tubes were incubated for 2 h at room temperature on a shaker. Each bead was then washed three times with 2 mL of demineralized water each time. The radioactivity was counted in a gamma counter for 60 s. The amount of S100 in the sample was then calculated using a standard curve prepared with calibrators with known concentrations of S100. S100 blood concentrations <0.5 µg/L were considered undetectable.

The data were analyzed by the Wilcoxon paired two-sided test and by linear regression analysis. A value of P <0.05 was considered as significant.

The concentrations of S100 in the blood of patients with favorable outcomes were undetectable or at the detection limit before sternotomy in patients successfully overcoming surgery and the postoperative period (Fig. 1 A), in accordance with previous reports using the same procedure to measure blood S100 (12). The concentrations increased during surgery, with the highest S100 values being detected at the end of CPB (time 2), when they were significantly higher (P <0.01, n = 11) than before surgery. Post-CPB S100 values decreased to concentrations not significantly different from those recorded before surgery. The two patients who died after surgery had the highest S100 concentrations during and after the surgical procedure and also exhibited detectable S100 concentrations before surgery.

View larger version (11K): [in this window] [in a new window]   Figure 1. S-100 blood concentrations in patients with favorable () and unfavorable () outcomes . (A) S100 concentrations at different surgical times as indicated in the text. The dotted line indicates the S100 detection limit (0.5 µg/L); and vertical lines are mean values and standard deviations, respectively, of S100 blood concentrations in patients with favorable outcomes only. (B) Correlation of S-100 blood concentrations at time 2 in patients with favorable () and unfavorable () outcomes with CPB duration. Linear regression analysis for all patients (dotted line: r = 0.94, P <O.001, n = 13) and for patients with favorable outcomes only (solid line: r = 0.89, P <0.001, n = 11) vs CPB duration.

The relationship between S100 concentrations at the end of CPB (time 2) and the duration of CPB is shown in Fig. 1B . A highly significant correlation was found between the duration of perfusion and the concentrations of S100 in the blood, regardless of whether patients with unfavorable outcomes were included (r = 0.93, P <0.001, n = 13) or excluded (r = 0.89, P <0.001, n = 11) from the statistical analysis.

Clinical parameters recorded during surgery remained within limits regarded as within the reference ranges during this type of procedure, and no overt neurological injury could be observed in surviving patients during the first week after surgery. There were no discernible clinical differences in the favorable outcomes of patients.

The present data on child patients are consistent with those reported in adults (8)(9)(10)(12). Therefore, it seems reasonable to assume that in children, when peculiar hemodynamic adaptive mechanisms in the brain are present (11), CPB is accompanied by at least a transient cerebral dysfunction, which has been recognized in adults (13)(14). Although the etiology of cerebral injury is known to be multifactorial and the release of S100 is compatible with flow-related brain microembolization, as recently hypothesized for adults (15), it is interesting that neurological disorders did not appear in the surviving patients examined in the present study; therefore, the leakage of this brain constituent indicates clinically undetectable neurological events. It is also noteworthy that neuron abnormalities detectable only at the electron-microscopic concentration, such as alterations in Golgi cisterns, have been observed in animals experimentally subjected to CPB (16). The possibility that increased permeability in the blood-brain barrier might play a role in increasing blood S100 concentrations must also be taken into account.

The especially high S100 concentrations observed in patients with unfavorable outcomes appear relevant. In particular, although the detection of S100 in the blood before surgery might be related to a series of factors difficult to evaluate, the possibility of preoperative clinically silent neurological dysfunctions in these patients must be taken into account because the appearance in the blood of the protein, which is usually undetectable in healthy children (12), is regarded as an indicator of distress in the nervous system (5). In light of the above considerations, blood S100 measurements might also be proposed for useful preoperative screening. Likewise, more extensive longitudinal studies of patients suffering postoperative neurological sequelae could reasonably offer indications on the usefulness of S100 measurements as prognostic indicators of brain distress or damage during CPB in child patients, as has been proposed recently for adult patients (9).

This work was partially supported by grants to Fabrizio Michetti from Consiglio Nazionale delle Ricerche and Ministero dell'Università e Ricerca Scientifica e Tecnologica. We thank Sangtec Medical, Bromma, Sweden, for supporting analysis kits.

Footnotes

fax 39 6 3051343, e-mail ibiis{at}rm.unicatt.it

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