Ann Thorac Surg 1996;62:1820-1824
© 1996 The Society of Thoracic Surgeons
Original Articles: Cardiovascular
Granulocyte Phagocytic Function Is Impaired During Cardiopulmonary Bypass
Kimikazu Hamano, MD,
Hiroshi Ito, MD,
Tomoe Katoh, MD,
Yoshihiko Fujimura, MD,
Hidetoshi Tsuboi, MD,
Kensuke Esato, MD
First Department of Surgery, Yamaguchi University, School of Medicine, Yamaguchi, Japan
Accepted for publication July 3, 1996.
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Abstract
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Background. The presence of impaired phagocytic function of the reticuloendothelial system after cardiac operations using cardiopulmonary bypass remains controversial.
Methods. In this study, the phagocytic function of granulocytes in 14 patients undergoing cardiac operations with cardiopulmonary bypass was examined using a chemiluminescence method. Seven patients with abdominal aortic aneurysms served as controls. Electron microscopy also was employed to evaluate morphologic changes.
Results. The 14 cardiac patients showed impaired phagocytic function from immediately after operation until 12 hours after the operation. This phagocytic function recovered within 24 hours. The 7 control patients showed no change in phagocytic function during or after the operation. Scanning electron microscopic examination of the cardiac patients' granulocytes revealed the loss of villi on cell surfaces immediately after operation. However, these villi were restored within 24 hours after the operation.
Conclusions. The phagocytic function of granulocytes was impaired in the early postoperative period in patients undergoing cardiopulmonary bypass, and this was probably due to the loss of villi on granulocyte surfaces.
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Introduction
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Although cardiopulmonary bypass (CPB) is essential to cardiac surgery, it has many drawbacks for the patient including complement activation and blood cell damage [1, 2]. Both cellular immunity [3], and humoral immunity are known to be depressed after CPB [4]. However, it remains controversial whether or not CPB impairs the function of the reticuloendothelial system (RES) [5–7]. The RES has two major components: phagocytic organs such as the spleen and liver, and phagocytic cells in the blood such as granulocytes. The phagocytic function of granulocytes in patients who are undergoing cardiac operations using CPB has not been fully investigated. In this study, this phagocytic function was examined in blood samples obtained during and after operations employing CPB using a chemiluminescence method [8]. Furthermore, morphologic changes in granulocytes were assessed in samples from several time points by scanning electron microscopy.
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Material and Methods
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Fourteen patients undergoing cardiac operations requiring CPB and 7 patients undergoing abdominal aortic reconstruction for abdominal aortic aneurysms (AAA) were studied after informed consent had been obtained and this experiment was reviewed by the Medical Ethics Committee of the Yamaguchi University School of Medicine. All patients were clinically stable upon entry into the study, and there was no evidence of infection or organ failure in any of the patients.
The characteristics and operative details of the 14 patients undergoing cardiac operations and 7 patients undergoing abdominal aortic aneurysmectomy are outlined in Table 1
. For the CPB circuit, a centrifugal pump (Bio-Console; Bio-Medicus, Inc, Eden Prarie, MN) and a membrane oxygenator (Capiox-E; Terumo Co, Tokyo, Japan) were used. The pump, circuit, and oxgenator were primed with 3 mL/kg of 20% mannitol, 3 mg/kg of betamethasone sodium phosphate (Rinnderon), Ringer's lactate solution, and heparinized blood (100 units per 100 mL) if required. The total priming volume was 1.8 L, with an ideal dilution rate of 20% to 25% and a perfusion index of 2.4 L•min-1•m-2 body surface area. An initial prebypass bolus dose of heparin (3 mg/kg) was infused to maintain an activated clotting time of more than 400 seconds during bypass. The operative procedures were performed under whole-body moderate hypothermia with rectal temperatures maintained between 27°C and 29°C. A potassium-based cold cardioplegic solution was intermittently administered through the aortic root, and topical cooling was applied to the heart. After CPB, protamine sulfate (same as the heparin dose) was administered.
Seven elective abdominal aortic reconstructions were performed for infrarenal aortic aneurysm. The abdominal aorta was exposed from the renal artery level to the origin of the external iliac arteries. After administration of heparin (0.5 mg/kg), the abdominal aorta and both iliac arteries were clamped. Aneurysmectomy was performed and the aorta was reconstructed with a Y-shaped woven Dacron prosthetic graft. In the AAA group, no steroids were administered.
Sample Collection
Blood samples for measuring the phagocytic function and morphologic examination were obtained via an arterial line that had been placed in the radial artery. If the arterial line was removed, blood samples were obtained from a femoral artery. Blood samples were collected preoperatively, immediately after the operation, 6, 12, and 24 hours after the operation, and 3, 5, and 7 days postoperatively. One-milliliter and 6-mL blood samples were collected in heparin-coated disposable syringes for measurement of the phagocytic function and morphologic examination, respectively.
White blood cell (WBC) counts and the number of granulocytes were measured using an automated cell counter (Sysmex K-1000; TOA Medical Electronics, Co, Ltd, Kobe, Japan) simultaneously for each collection time.
Measurement of the Phagocytic Function
Phagocytic function was measured using a chemiluminescence method [8]. Two hundred microliters of whole blood, 1.65 mL of Hanks' balanced salt solution, and 100 µL of diluted luminol (50 µmol/L) (5-amino-2,3-dihydro-1,4-phthalazinedione) (Sigma, St. Louis, MO) were mixed in a test tube and incubated for 15 minutes at 37°C. Fifty microliters of zymosan solution (40 mg of zymosan [Tokyokasei, Tokyo, Japan] in 1 mL of Hanks' balanced salt solution) was added and incubated for 30 minutes at 37°C. Chemiluminescence was measured using a luminescence reader (Aloka Co, Ltd, Tokyo, Japan). The peak chemiluminescence (counts/min) was divided by the number of granulocytes and considered the phagocytic index to correct for the changing number of granulocytes.
Morphologic Changes in the Granulocytes
Granulocyte purification was performed according to the following method [9]. Three milliliters of Histopaque-1119 (Sigma) and 3 mL of Histopaque-1077 (Sigma) were layered on 6 mL of whole blood. The tube was centrifuged at 700 g for 30 minutes at room temperature. Because the granulocytes should have deposited in the interface between Histopaque-1119 and Histopaque-1077, this layer was collected. The granulocytes were then washed with phosphate-buffered saline solution three times to remove the Histopaque. For the morphologic examination, granulocytes were collected directly into 2% paraformaldehyde/0.2% glutaraldehyde in 0.1 mol/L cacodylate buffer (pH 7.4). Granulocytes were fixed for 1 hour at room temperature, rinsed three times with 0.1 mol/L cacodylate buffer, placed on poly-L-lysine-coated (1 mg/mL) coverslips, and postfixed with 2% osmic acid for 2 hours. They were then processed as previously described [10] and examined with a JEOL T-300 scanning electron microscope (Tokyo, Japan).
Statistics
The WBC and granulocyte counts are presented as the mean ± the standard deviation, and the phagocytic indices of the blood are presented as the mean ± the standard error of the mean. The paired Student's t test was used to compare changes over time. A p value of less than 0.05 was considered significant.
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Results
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Changes in White Blood Cell Counts
The changes in WBC counts in both groups are presented in Figure 1. There were no significant differences between the AAA (n = 7) and the CPB groups (n = 14) preoperatively. In the AAA group, the WBC count peaked immediately postoperatively (12,700 ± 4,623/µL). However, in the CPB group, the WBC count gradually increased, peaking 3 days after the operation. There were significant differences (p < 0.05) in WBC counts between the two groups immediately postoperatively and 6 hours after the operation.
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Fig 1. . Changes in the white blood cell counts in both groups. In the abdominal aortic aneurysm group (squares), white blood cell counts peaked immediately postoperatively. In the cardiopulmonary bypass group (circles), white blood cell counts gradually increased, peaking 3 days postoperatively. There were significant differences between the two groups immediately postoperatively and 6 hours after the operation (*p < 0.05). (Postop = immediately postoperative; Preop = preoperative.)
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Changes in Granulocyte Counts
The changes in granulocyte counts in both groups are presented in Figure 2, and demonstrate a pattern similar to the changes in WBC counts in both groups. In the AAA group (n = 7), the granulocyte count peaked 6 hours postoperatively. However, in the CPB group (n = 14), the peak granulocyte count was delayed until 24 hours after the operation. There were significant differences in granulocyte counts between two groups immediately postoperatively and 6 hours after the operation.
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Fig 2. . Changes in the granulocyte counts in both groups. In the abdominal aortic aneurysm group (squares), granulocyte counts increased postoperatively with a peak at 6 hours. In the cardiopulmonary bypass group (circles), granulocyte counts increased gradually with a peak at 24 hours postoperatively. There were significant differences between the two groups immediately postoperatively and 6 hours after the operation (*p < 0.05). (Postop = immediately postoperative; Preop = preoperative.)
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Changes in the Phagocytic Index
Preoperatively, level of the phagocytic index of the AAA group (n = 7) (29.1 ± 7.2 counts/min) did not significantly differ from that of the CPB group (n = 14) (29.4 ± 4.8 counts/min). Furthermore, the phagocytic index did not significantly change throughout the study in the AAA group (Fig 3).
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Fig 3. . Changes in the phagocytic index. In the abdominal aortic aneurysm group (squares), the phagocytic index did not significantly change before, during, or after the operation. In the cardiopulmonary bypass group (circles), the phagocytic index from immediately postoperatively to 12 hours after the operation was significantly reduced compared with the preoperative level (*p < 0.01). (CPM = counts per minute; Postop = immediately postoperative; Preop = preoperative.)
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In the CPB group, however, the phagocytic index immediately postoperatively (11.2 ± 3.1 counts/min) was reduced significantly compared with the preoperative level (29.4 ± 4.8 counts/min; p < 0.01). This decreased phagocytic index was maintained until 12 hours after the operation with subsequent recovery by 24 hours postoperatively. The phagocytic index did not significantly change from 24 hours to 7 days after the operation compared with the preoperative level.
Morphologic Changes in Granulocytes in the Cardiopulmonary Bypass Group
To determine the cause of the impaired phagocytic function, we examined morphologic changes in granulocytes obtained during and after the operation in the CPB group. Scanning micrographs of granulocytes obtained immediately postoperatively revealed that the villi of the granulocytes were heavily damaged and not visible in almost all cases (Fig 4). However, the granulocyte villi had recovered by 24 hours after CPB, and their appearances on granulocyte cell surfaces did not change for the additional 7 days after the operation. The time course of those morphologic changes correlated well with the decrease in the phagocytic function (index) of the granulocytes. The morphologic appearance of granulocyte villi in the AAA group did not show a significant change postoperatively.
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Fig 4. . Typical scanning electron micrographs of the granulocytes before and after operation in the cardiopulmonary bypass group. (A) Preoperatively, the granulocytes appeared normal. (B) Immediately postoperatively, the villi of the granulocytes had disappeared. (C) By 24 hours after the operation, the villi of the granulocytes had recovered. (x10,000 before 11% reduction.)
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Comment
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The RES is an important component in the body's defense. A compromise of RES function is associated with serious infections. Reticuloendothelial system function previously has been evaluated by measuring plasma fibronectin level, which is a major opsonic protein in the RES [11–13]. The authors of those reports concluded that RES function was impaired after CPB because the plasma fibronectin level was reduced. However, direct measurement of RES function has produced varying results [5–7,14]. Although Subramanian and associates [14] and Silva and colleagues [5] have shown experimental and clinical evidence of impaired RES function, respectively, other investigators have reported normal RES function after CPB [6, 7]. Therefore, there is still controversy regarding RES function after CPB.
The RES has two major components: phagocytic organs (liver, spleen) and phagocytic cells in the circulating blood. In this study, we focused on the phagocytic function of granulocytes in the circulating blood. We used whole blood because some investigators [8] have reported that using whole blood to measure phagocytic function is reliable.
The changes in WBC and granulocyte counts showed a different pattern between the AAA and the CPB groups. After a major operation inflammation occurs in patients due to operative tissue damage. As part of this process the numbers of WBCs, including granulocytes, are increased by cytokines [15]. As expected, total WBC and granulocyte counts increased postoperatively in the AAA group. However, the CPB group showed a different pattern. It has been reported that WBC counts peak at 12,000 to 24,000/µL at 24 to 48 hours postoperatively [16]. In our study, the WBC count in the CPB group peaked 3 days after the operation. This is similar to the results of previous reports. Steroids are commonly used in cardiac operations employing CPB, because they improve tissue perfusion and prevent increases in extracellular water content. Steroids also exert an antiinflammatory effect. Thus, the difference in WBC and granulocyte counts between the two groups might be due to the use of steroids in the CPB group.
Our results also suggested that the phagocytic index, measured via a chemiluminescence method, was reduced after CPB, indicative of impaired phagocytic function. This dysfunction continued until 12 hours after CPB. Subsequently, the phagocytic function recovered to normal. As a control, the phagocytic function of granulocytes in patients with AAAs also was measured, but no significant changes were observed during the study in this group.
Next, we tried to determine the cause of the impairment in phagocytic function in the CPB group. During CPB, the circulating cells contact artificial devices, such as the oxgenator and the pump circuit. Therefore, we examined the morphologic changes in the granulocytes, which mainly carry out the phagocytic function in the circulating blood. Scanning electron microscopic examination revealed a dramatic change in the granulocytes after CPB. The villi of the granulocytes almost completely disappeared after CPB, probably due to the mechanical damage caused by CPB. However, the villi recovered 24 hours after CPB. There are two possible ways for the villi to reappear. One is their reexpression on circulating granulocytes, and the other is translocation of new granuloctes from the bone marrow. These morphologic changes correlated well with the changes in phagocytic function. Steroids used in priming the CPB circuit might suppress phagocytic function. Villi are necessary for granulocytes to carry out phagocytosis [17, 18], serving as sensors for contacting a foreign body as well as initial stages of pseudopods that will surround the foreign body and engulf it [17]. Loss of the villi, therefore, impairs phagocytic function and may serve as the basis for steroid-associated impairment of phagocytosis. Mannitol exposure also might have influenced the morphologic changes of granulocytes. From scanning electron micrographs, the mean size of the granulocytes was determined to be larger after the operation. This means that activation of the granulocytes had occurred. In the early postoperative period, the granulocytes exhibited reduced phagocytic function, despite the occurrence of intracellular activation as described previously [19]. In the CPB group, protamine sulfate was added to diminish the effect of heparin. Protamine sulfate has no reported effect on phagocytic function.
Currently some cases of coronary artery bypass grafting are performed without CPB [20, 21]. This approach is able to avoid the several adverse effects of CPB. These results also recommend coronary artery bypass grafting without CPB from the point of view of the immune defense. Although CPB is essential for many cardiac operations, we must keep trying to decrease the CPB time to decrease the several adverse effects of CPB. The infection rate in patients recovering from CPB might not differ significantly from that of patients having other major operations, but surgeons need to be aware of the patients' impaired immunity after CPB and the mechanisms likely to contribute to it.
In this study we measured the phagocytic function of granulocytes in the circulating blood and showed impaired phagocytic function early after CPB. Furthermore, morphologic examination of the granulocytes revealed that the impairment in phagocytic function correlated with the disappearance of villi from the cell surfaces.
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Footnotes
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Address reprint requests to Dr Hamano, First Department of Surgery, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan 755.
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References
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- Chenoweth DE, Cooper SW, Hugli TE, et al. Complement activation during cardiopulmonary bypass. N Engl J Med 1981;304:497–503.[Abstract]
- Kusserow B, Larrow R, Nicols J. Perfusion and surface-induced injury in leukocytes. Fed Proc 1971;30:1516–20.[Medline]
- Tajima K, Yamanoto F, Kawazoe K, et al. Cardiopulmonary bypass and cellular immunity: changes in lymphocyte subsets and natural killer cell activity. Ann Thorac Surg 1993;55:625–30.
- Van Velzen-Blad H, Dijkstra YJ, Schurink GA, et al. Cardiopulmonary bypass and host defence functions in human beings: I. Serum levels and role of immunoglobulins and complement in phagocytosis. Ann Thorac Surg 1985;39:207–11.[Abstract]
- Silva J Jr, Hoeksema H, Fekety FR Jr. Transient defects in phagocytic functions during cardiopulmonary bypass. J Thorac Cardiovasc Surg 1973;67:175–83.[Medline]
- Schildt B, Berghem L, Holm G, et al. Influence of cardiopulmonary bypass on some host defense functions in man. Scand J Thorac Cardiovasc Surg 1980;14:207–11.[Medline]
- Shimono T, Nose Y, Yada I, et al. Is it true that cardiac operations with cardiopulmonary bypass impair the reticuloendothelial system? Ann Thorac Surg 1994;58:1059–63.[Abstract]
- Tono-Oka T, Ueno N, Matsumoto T, Ohkawa M, Matsumoto S. Chemiluminescence of whole blood. Clin Immunol Immunopathol 1983;26:66–75.[Medline]
- English D, Andersen BR. Single-step separation of red blood cells, granulocytes and mononuclear leukocytes on discontinuous density gradient of Ficoll Hypaque. J Immunol Methods 1974;5:249–52.[Medline]
- Yamaguchi K, Fujikura Y, Kuniki H, et al. Immunoelectron microscopic localization of cell surface antigens on rat hepatocytes detected with monoclonal antibodies (HAM2 and HAM4). Cell Struct Funct 1991;16:303–13.[Medline]
- Snyder EL, Barash PG, Mosher DF, Walter SD. Plasma fibronectin level and clinical status in cardiac surgery patients. J Lab Clin Med 1983;102:881–9.[Medline]
- Pourrat E, Sie PM, Desrez X, et al. Changes in plasma fibronectin levels after cardiac and pulmonary surgery: role of cardiopulmonary bypass. Scand J Thorac Cardiovasc Surg 1985;19:63–7.[Medline]
- Charash WE, Foster ED, Saba TM, Dayton C, Cho E. Plasma fibronectin levels during cardiopulmonary bypass. J Appl Physiol 1990;69:1644–50.[Abstract/Free Full Text]
- Subramanian V, McLeod J, Gans H. Effect of extracorporeal circulation on reticuloendothelial function. I. Experimental evidence for impaired reticuloendotherial function following cardiopulmonary bypass in rats. Surgery 1968;64:775–84.[Medline]
- Cruickshank AH. Inflammation. In: McFarland J, ed. Basic clinical surgery for nurses and medical students. 2nd ed. London: Butterworths, 1980:161–l78.
- Kirklin JW, Barratt-Boyes BG, ed. Cardiac surgery. 2nd ed. New York: Churchill Livingstone, 1993:85–9.
- Moore PL, Bank HL, Brissie NT, Spicer SS. Phagocytosis of bacteria by polymorphonuclear leukocytes. J Cell Biol 1978;76:158–74.[Abstract/Free Full Text]
- Tokunaga M, Tokunaga J, Niimi M. Leukocyte and macrophage movements under phagocytosis. In: Tanaka K, Fujita T, eds. Scanning electron microscopy in cell biology and medicine. Amsterdam: Excerpta Medica, 1981:13–o22.
- Riegel W, Spillner G, Schlosser V, Horl W. Plasma levels of main granulocyte components during cardiopulmonary bypass. J Thorac Cardiovasc Surg 1988;95:1014–9.[Abstract]
- Benetti FJ, Naselli G, Wood M, et al. Direct coronary artery surgery with saphenous bypass without either cardiopulmonary bypass or cardiac arrest. J Cardiovasc Surg 1985;26:217–22.[Medline]
- Pfister AJ, Zaki MS, Garcia JM, et al. Coronary artery bypass without cardiopulmonary bypass. Ann Thorac Surg 1992;54:1085–92.[Abstract]
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Abstract
Introduction
