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CARDIOVASCULAR ANESTHESIA

Are Leukocytes in Salvaged Washed Autologous Blood Harmful for the Recipient? The Results of a Pilot Study

Petra Innerhofer, MD^*, Franz J. Wiedermann, MD^*, Werner Tiefenthaler, MD^*, Wolfgang Schobersberger, MD^*, Anton Klingler, PhD, Corinna Velik-Salchner, MD^*, Elgar Oswald, MD^*, Erwin Salner, MD^*, Eveline Irschick, PhD, and Gabriele Kühbacher, MD^*

*Department of Anesthesia and Critical Care Medicine, Theoretical Surgery Unit, Department of General Surgery, and Department of Ophthalmology, Immunological Laboratory, The Leopold-Franzens University of Innsbruck, Innsbruck, Austria

Address correspondence and reprint requests to Petra Innerhofer, MD, Department of Anesthesia and Critical Care Medicine, The Leopold-Franzens University of Innsbruck, Anichstr. 35, A-6020 Innsbruck, Austria. Address e-mail to Petra.Innerhofer{at}uibk.ac.at

	Abstract

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To explore whether polymorphonuclear leukocytes (PMNL) are activated to the priming threshold through intraoperative blood salvage, and are thus able to induce endothelial damage, we investigated chemotactic response (n = 20) and respiratory burst (RB; n = 20) of PMNL without (basal respiratory burst, bPMNL-RB) and after in vitro stimulation with formyl-Met-Leu-Phe (fMLP-RB) and phorbol myristate acetate (PMA-RB). Blood was processed with a continuous autotransfusion device (CATS). Heparin (Heparin group) and sodium citrate (Citrate group) were used alternately as an anticoagulant for each half of the chemotaxis and RB studies. Comparison of measurements from the processed autologous erythrocyte concentrates (paEC) to pre- and intraoperative arterial blood samples showed no statistically significant difference for any test of PMNL functional responses in an orthopedic patient population. Analysis of intraindividual changes demonstrated a significantly increased bPMNL-RB (both groups, P = 0.0032; Heparin group, P = 0.0098), fMLP-RB (both groups, P = 0.0484; Citrate group, P = 0.0371), and PMA-RB (Citrate group, P = 0.002) in the paEC compared with intraoperative arterial samples, whereas the chemotactic response did not change. Nevertheless, median values of all RB measurements in the paEC were within the range of pre- and intraoperative values, indicating that PMNLs contained in the paEC are neither impaired nor activated to the priming threshold. The results confirm the clinical experience that intraoperative blood salvage is safe to use during major orthopedic surgery and questions the beneficial effect of special leukocyte-removing filters.

IMPLICATIONS: Leukocytes contained in intraoperatively processed autologous blood using either heparin or citrate were not activated to the threshold that is associated with leukocyte-mediated tissue injury. This study raises the question of the necessity of special leukocyte filters for the transfusion of intraoperatively salvaged blood.

	Introduction

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Intraoperative blood salvage (IBS) has become increasingly common in major surgery; it provides rapidly available fresh red cells for autologous transfusion. The use of salvaged blood avoids the risks of disease transmission, red cell storage lesion, clerical error, and immunomodulation—all possible side effects associated with the transfusion of stored blood components (1–3). However, because of reports of increased concentrations of complement factors, lipid mediators, and increased expression of leukocyte adhesion molecules in salvaged blood, concerns about the safety of processed autologous blood remain (4–6). Activated polymorphonuclear leukocytes (PMNL) may induce endothelial damage and increase vascular permeability, coagulopathy, and severe pulmonary dysfunction, resulting in the so-called salvaged cell syndrome (7). However, an association between IBS and acute lung injury has been demonstrated only in an animal model (7). In patients receiving considerable amounts of salvaged blood, the development of postoperative acute respiratory distress syndrome is a complex clinical entity, with the influences of other etiological factors such as tissue trauma, blood loss, compromised microcirculation, and exposure to multiple transfusions being indistinguishable from the effects provoked by activated leukocytes contained in salvaged blood. Although clinical experience has shown excellent safety of IBS (8) and only two studies reported contradictory PMNL functional responses associated with IBS (9,10), some manufacturers recommend the use of special blood filters for retransfusion of processed autologous blood to eliminate these possibly harmful leukocytes.

The process of PMNL priming is critical for PMNL-mediated tissue injury. Primed PMNLs exhibit up to a 20-fold increase in superoxide anion generation, undergo polarization, shape change, and show modulated adhesion and migration (11). To explore whether IBS is associated with activation of PMNL to the priming threshold, we determined the chemotactic response (CT) and the respiratory burst of PMNL, two steps in the nonspecific immune response that have been established as valuable tools in investigating leukocyte function in patients under various clinical conditions (12–14). Because heparin or sodium citrate is required as an anticoagulant to prevent clotting of the salvaged blood, both types of anticoagulant solutions were used alternately.

	Methods

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The protocol for this study was approved by the Ethics Committee of the Leopold-Franzens University of Innsbruck, and informed consent was obtained from each patient. Forty consecutive patients undergoing major orthopedic procedures (surgery of the spine, revision joint replacement, or tumor surgery) for which IBS is used routinely were enrolled in the nonrandomized explorative pilot study of PMNL functional responses. To facilitate the immediate analysis of blood samples, the study was conducted in two subsequent parts: the chemotaxis study (n = 20) and the respiratory burst study (n = 20). Exclusion criteria were renal insufficiency, major hematologic and coagulation disorders, local and systemic infectious disease, preoperative radiotherapy, premedication with corticosteroids, and other immunosuppressive drugs.

All patients received standardized general anesthesia with thiopentone sodium (thiopental), fentanyl (Janssen-Cilag, Vienna, Austria), rocuronium (Esmeron^®; Organon-Teknika, Netherlands), and inhaled sevoflurane (Sevorane^®) in an air/oxygen mixture. All patients were actively warmed with fluid warmers (Biegler Biotest Pharmazeutika, Vienna, Austria) and a convective warming system (Bair Hugger Augustine Medical, Eden Prairie, MN). Monitoring during surgical intervention included invasive blood pressure measurement, central venous pressure measurement, electrocardiogram, pulse oximetry, capnometry, and urine output (urinary bladder catheter). During surgery, the continuous autotransfusion system (CATS; Fresenius, Frankfurt, Germany) was used to salvage and process red blood cells. This autotransfusion system has been described in detail elsewhere (15,16). In brief, contrary to systems using centrifuge bowls, the separating and washing process occurs continuously with a disk-shaped separation chamber (representing a blood channel in the shape of a double spiral) and three pumps for blood intake, erythrocyte concentrate outlet, and washing solution. The automated separation and washing process is independent of the volume of salvaged blood processed, and the hematocrit (Hct) of the resulting erythrocyte concentrate is constant. For the first 10 procedures of the chemotaxis and respiratory burst study, heparin (Heparin group, 30,000 IU/L NaCl) was used as anticoagulant, and sodium citrate (Citrate group, trisodium citrate 46.7% 60 mL/L NaCl) was used for the subsequent 10 consecutive procedures. The collecting reservoir was primed with 100 mL of anticoagulation solution at the start of surgery, followed by intraoperative rates of 3 mL/min for heparin solution and 2 mL/min for citrate-containing solution.

CT and respiratory burst of PMNL were determined from the patients’ arterial blood before surgery for comparison of baseline conditions of patients in the Heparin and Citrate groups. A second sample was drawn from the patients’ arterial blood during the operation, because changes in PMNL functional responses occur with surgical trauma and anesthesia. This sample was drawn before transfusion of any blood products, and at the same time, the continuous autotransfusion device started to process the collected wound blood (1000 mL on average) and a sample was drawn from the resulting autologous erythrocyte concentrate (paEC). By using standard laboratory methods (Coulter Counter STKS; Beckmann Coulter Inc., Miami, FL), blood cell count was measured before surgery and in the paEC.

Methods for PMNL isolation and assays for CT and respiratory burst have been described in detail (17,18). In brief, to minimize artificial changes in leukocyte functional responses, blood samples were drawn from arterial catheters into EDTA-containing plastic tubes and stored at room temperature for a maximum of 2 h. PMNLs were separated from whole blood by means of discontinuous density gradient centrifugation with a Percoll device (Sigma, St. Louis, MO). In vitromigration was assayed with a microchemotaxis chamber (Neuroprobe, Bethesda, MD). The CT of PMNL is expressed as the chemotaxis index, namely, the ratio between the microscopically measured distance of directed (interleukin-8) and undirected (buffer) migration of PMNL into the nitrocellulose filter of the chemotaxis chamber.

For the respiratory burst assay, separated PMNL were concentrated to 2 x 10⁵ PMNL per milliliter. The basal respiratory burst activity of neutrophils and the activity after stimulation with formyl-Met-Leu-Phe (fMLP) and phorbol myristate acetate (PMA) were measured fluorometrically by use of dichlorofluorescein diacetate (DCFH-DA). This assay is based on the oxidation of nonfluorescent DCFH-DA to highly fluorescent 2'7'-dichlorofluorescein (DCF), both intracellularly and extracellularly. The fluorescence activity was determined at 485-nm excitation and 530-nm emission wavelengths by using the CytoFluor 2350 fluorescence measurement system (Millipore Corp., Bedford, MA).

Measurements of CT and respiratory burst of PMNL were performed in quadruplicate for each patient and time point. Data collected from patients included age, type of surgical procedure, duration of surgery, total amount of blood collected in the reservoir, and total amount of retransfused salvaged red cells.

The study was intended as an explorative study without primary comparison of heparin with sodium citrate. We therefore did not calculate probe size, nor did we randomize the use of heparin or sodium citrate. Data in tables are given as median (range), and data in figures are expressed in box plots: 10th, 25th, 50th (median), 75th, and 90th percentiles, as well as points above the 90th and below the 10th percentile. Differences within groups were calculated with the Wilcoxon-Mann-Whitney test for paired samples, and the Wilcoxon’s rank test was used for comparison of baseline values between groups. To analyze the influence of age on measurements of leukocyte functional response, a linear regression model has been used. A P value <0.05 was considered statistically significant.

	Results

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Patients experienced similar surgical trauma, as shown in Table 1, and were comparable, with the exception that in the Heparin group of the chemotaxis study, patients were younger and body weight was slightly less. Linear regression, however, demonstrated no influence of age or body weight on leukocyte functional responses (CT, P = 0.8247). Preoperative measurements of blood cell count, CT, basal PMNL respiratory burst (bPMNL-RB), and respiratory burst after stimulation with PMA (PMA-RB) and fMLP (fMLP-RB) were similar in patients in the Heparin and Citrate groups (Tables 1 and 2).

View larger version (18K): [in this window] [in a new window]   Figure 1. The chemotaxis response (CT) of leukocytes in the processed autologous erythrocyte concentrate (paEC) blood did not differ from that of the native patient leukocytes (preoperative and intraoperative arterial samples), and no difference was seen when heparin (CT-HEP) or citrate (CT-CIT) was used as anticoagulant for the salvaged blood.

View larger version (30K): [in this window] [in a new window]   Figure 2. (a–c) Fluorometrically measured respiratory burst (RB) of polymorphonuclear leukocytes (PMNL) without (a) (bPMNL-RB) and after in vitrostimulation with phorbol myristate acetate (PMA) (b) (PMA-RB) and formyl-Met-Leu-Phe (fMLP) (c) (fMLP-RB) is shown for patients’ pre- and intraoperative arterial blood and for the sample drawn from the processed autologous erythrocyte concentrate (paEC) by using the continuous autotransfusion device and heparin (HEP) or citrate (CIT) as anticoagulant. Median values of PMNL respiratory burst in the paEC were similar to those measured in the patient before and after surgery, regardless of whether heparin or citrate was used as an anticoagulant.

Although these differences were statistically sig- nificant, the median values of all PMNL functional responses in the paEC were within the range of preoperative values (Table 2). Additional maximal measured values were largely less than pre- and intraoperative maximal values, and only in 3 of 20 measurements (one measurement each for bPMNL-RB, PMA-RB, and fMLP-RB) was RB above the maximum values measured in the patient. As a second result, we observed a comparable Hct of the paEC processed with heparin as compared with citrate (Heparin groups, 69.6% ± 7.8%; Citrate groups, 66.8% ± 8.3%), but there were significant differences in leukocyte and platelet counts. Leukocyte counts in the Heparin groups were 3.6 ± 1.7 x 10⁹/L and in the Citrate groups, 6.7 ± 2.5 x 10⁹/L; platelet counts in the Heparin groups were 19.2 ± 15.9 x 10⁹/L and in the Citrate groups, 46 ± 42.1 x 10⁹/L.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This is the first study to search for a potentially harmful activation of PMNL by IBS by using heparin or sodium citrate as an anticoagulant. The main finding of our study is that leukocytes contained in processed autologous blood are neither activated to the priming threshold nor impaired. Median values of chemotaxis and RB assays were within the range measured in the patients’ native blood. Furthermore, results were independent of whether heparin or citrate was used as the anticoagulant solution.

PMNLs are a first line of defense, and various stimuli initiate a complex cascade that results in adhesion, migration, and phagocytosis of microorganisms and inflammatory debris. Concomitantly PMNLs increase their oxygen consumption and generate superoxide anions and hydrogen peroxide, which are antimicrobial but may also cause damage to the surrounding tissues (19). The induction of endothelial injury by PMNL, leading to acute lung injury, strongly depends on a prior priming process that prompts a potentiated response of PMNL to an activating stimulus (11). A comparison of measurements from the patients’ pre- and intraoperative native blood and those obtained from the paEC showed no significant difference for any test of PMNL functional responses. This general comparison of the three measurements might be inconclusive, because surgical trauma and anesthesia alone can influence leukocyte responsiveness. Indeed, we found that chemotaxis increased during surgery, whereas bPMNL-RB decreased and fMLP-RB showed a trend toward a decrease. Therefore, the analysis of intraindividual differences, intraoperative arterial to paEC (Table 3) seems of major importance in determining the influence of the autotransfusion process on PMNL response. Measurements in the paEC showed an unchanged CT, and those of basal and stimulated RB were significantly increased compared with the intraoperative sample from these patients. However, all median values of CT and RB measured in the paEC were within the range of pre- and intraoperative values. In 3 of 20 measurements (one measurement each for bPMNL-RB, PMA-RB, and fMLP-RB), RB was above the maximum values measured in the patient. Considering that primed PMNLs exhibit a superoxide anion release, which may be enhanced up to 20-fold, even these maximum values were only moderately increased outside the pre- and intraoperative range, meaning that retransfusion of such PMNL should not be harmful for the recipient.

Perttilä et al. (9) studied the activity of leukocytes in prepared autologous blood during cardiac surgery by using a discontinuous autotransfusion system in which RB was determined by chemiluminescence. No significant differences were reported between mea-surements from the patient and those from the retransfusion blood. Although neutrophil activity in the processed blood was 30% higher in 4 of 10 patients only, the authors recommended that processed blood not be retransfused when reperfusion injury can be expected. In contrast to the results of Perttilä et al. and ours, Seidel et al. (10) reported an even more significantly impaired CT and superoxide generation of PMNL after IBS as compared with PMNL from healthy volunteers.

Thus, concerns about the safety of IBS are sustained solely by data from one animal model (7) and the results of three studies (4–6) reporting increased measurements of complement factors, leukotriene B4, platelet-activating factor, and ß-integrins in blood processed with discontinuous systems and with heparin as anticoagulant. However, performance of IBS in the animal model markedly differs from clinical application of IBS, and therefore results might be due to a particular study design (7). Furthermore, measurements of PMNL-associated mediators (4–6) depend on the Hct of the processed blood and the number of leukocytes contained in the samples, both of which might be influenced by several variables other than use of IBS (20).

This study shows the consequences of IBS during clinical practice. To eliminate possible confounding factors, patients underwent standardized anesthesiologic management, and samples were drawn before transfusion of any blood product. Moreover, we focused on functional tests of PMNL, whereby results are independent of Hct and leukocyte count in blood samples because PMNLs were separated and concentrated to a fixed number until assayed.

It was not our intention to clarify the question of the ideal anticoagulant solution during IBS. Nevertheless, data from the animal model (7) suggest that citrate is preferable because it minimizes concentrations of Ca²⁺. Consequently, the receptor-independent pathway of leukocyte activation (PMA-RB) should be impaired. We observed no differences in PMA-RB between groups. It is interesting to note that in the Citrate group, PMA-RB was significantly increased in the paEC as compared with pre- and intraoperative measurements from the patient. Standard deviation of PMA-RB measurements, however, was much greater in the Heparin group than in the Citrate group.

As a second result, we observed significantly lower counts of platelets and leukocytes in the processed autologous blood from patients in the Heparin groups. We speculate that heparin promotes PMNL platelet interactions, leading to formation of PMNL platelet complexes (21–24) that are removed by the washing process.

The limitations of our study are the nonrandomized study design and the fact that patients in the Heparin group of the chemotaxis study were younger compared with those in the Citrate group. However, no intervention occurred in the patients that were different, the same autotransfusion device was used for all procedures, heparin and citrate were routinely used for anticoagulation of the salvaged blood, and baseline measurements of PMNL functional responses showed no significant differences between groups. Moreover, linear regression analysis showed no influence of demographic variables on PMNL functional responses.

In conclusion, our data show that IBS using the continuous autotransfusion device and heparin- or citrate-containing solution as anticoagulant causes neither impairment nor priming of PMNL, which would induce endothelial damage. Our data raise the question whether special leukocyte filters, which also increase costs and impair flow conditions during retransfusion, are necessary to enhance the safety of IBS during major orthopedic surgery. Data from a larger randomized study are needed to answer the questions of the ideal anticoagulant during IBS and the clinical relevance of decreased counts of leukocytes and platelets in the presence of heparin.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (9) Citing Articles via Google Scholar Google Scholar Articles by Innerhofer, P. Articles by Kühbacher, G. Search for Related Content PubMed PubMed Citation Articles by Innerhofer, P. Articles by Kühbacher, G. Related Collections Cardiovascular Blood