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*Department of Cardiothoracic Anesthesia and Intensive Care, Department of Cardiothoracic Surgery, Department of Immunology and Transfusion Medicine, and Department of Medical Biochemistry, St. Olav University Hospital, and ||Unit for Applied Clinical Research, ¶Department of Circulation and Medical Imaging, and #Department of Laboratory Medicine, Children's and Women's Health, Norwegian University of Science and Technology, Trondheim, Norway
Address correspondence and reprint requests to Hilde Pleym, MD, St. Elisabeth Department of Cardiothoracic Surgery, Hans Nissens gate 3, N-7018 Trondheim, Norway. Address e-mail to hilde.pleym{at}stolav.no.
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Abstract |
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Introduction |
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In a study of perioperative hemostasis during major abdominal surgery, Boldt et al. (7) showed that compared with younger patients, elderly patients had continuing activation of the coagulation system, with increased prothrombin activation, thrombin generation, and fibrinolytic activity before surgery. These changes may imbalance the hemostatic system and result in increased postoperative bleeding. In cardiac surgery, thromboelastography (TEG®) has been used to investigate whether hemostasis differed between elderly and younger patients (8). The TEG® results showed that elderly cardiac surgical patients had signs of a moderate coagulopathy perioperatively. The authors concluded that the exact reasons for the observed impairment of coagulation measured by TEG® remain to be elucidated.
The aim of the present study was to explore and describe the perioperative hemostatic process in elderly compared with younger patients undergoing coronary artery bypass grafting (CABG). To this end, various factors involved in coagulation, fibrinolysis, and platelet activation were investigated in a group of elderly and a group of younger patients preoperatively and postoperatively.
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Methods |
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All patients had first-time CABG with the use of the left internal thoracic artery and saphenous vein bypass grafts. Enrolled patients were assigned to one of the four attending surgeons on a rotational basis to ensure that each surgeon operated on an equal number of elderly and younger patients. In addition, patients were matched in pairs, and the same surgeon operated on both patients in a pair. The patients were matched for expected number of bypasses to be performed to ensure that the surgical time, cardiopulmonary bypass (CPB) time, and cross-clamp time would be approximately equal in each pair of patients. Antifibrinolytic drugs (aprotinin, tranexamic acid, 
-aminocaproic acid) or desmopressin were not given. CPB was used in all cases, and all patients were anesthetized with a standard technique based on a combination of fentanyl and isoflurane. Before CPB, heparin 300 U/kg (Leo, Copenhagen, Denmark) was given through a central venous catheter to achieve a kaolin activated clotting time (ACT) (Medtronic Blood Management, Parker, CO) of >480 s. The ACT was measured in duplicate and the mean value was recorded. Additional heparin was given when needed to keep the ACT above the target. During CPB the ACT was monitored every 20 min. The perfusion circuit was primed with 1800 mL of Ringer's acetate solution to which 7500 U of heparin was added. A membrane oxygenator without heparin coating was used. Cold antegrade crystalloid or blood cardioplegia and moderate hypothermia to 34°C were used during CPB. Cardiotomy suction was used while the patients were fully anticoagulated, and the blood was returned to the patients without centrifugation. The patients were warmed to a rectal temperature of at least 36°C before termination of CPB. After CPB, protamine sulfate 1 mg for every 100 U of previously administered heparin (prime heparin not included) was given to achieve an ACT within 10% of the baseline value. Additional doses of protamine were given when necessary. Blood remaining in the CPB circuit was collected and transfused to the patients.
At the end of surgery the patients were transferred to the intensive care unit. All patients were sedated with repeated doses of midazolam 1–2 mg or a small-dose infusion of propofol (0.5–1 mg · kg–1 · h–1) until tracheal extubation. Postoperatively, blood from the mediastinal and pleural drains was collected in a sterile cardiotomy reservoir (Cardiotomy Reservoir, filtered; Sorin Biomedica UK, LTD, Harrogate, UK) and autotransfused until bleeding was <20 mL/h, but for a maximum of 8 h. All patients were autotransfused shed blood. Transfusions of packed red blood cells were given when the blood hemoglobin concentration was <8.5 g/dL. Increased postoperative bleeding was treated with infusions of fresh-frozen plasma and/or platelets. The final decision to give platelets or fresh-frozen plasma was left to the attending physician, but the treatment was only considered when patients had a persistent postoperative bleeding of >200 mL/h.
The primary study outcome was the preoperative and postoperative measurements of variables indicative of hemostatic function. Blood samples were drawn from the arterial catheter preoperatively before induction of anesthesia, at 30 min and 3 h after the end of surgery, and on the morning of the first postoperative day, approximately 20 h after surgery. Routine coagulation variables were analyzed consecutively by standard methods and included platelet counts, INR, APTT, and fibrinogen. Prothrombin fragment 1 + 2 (F1 + 2) and thrombin-antithrombin complex (TAT) were measured as indicators for increased thrombin formation, and TAT and antithrombin were measured also as indicators of increased inhibitor consumption. d-dimer was measured as an indicator of fibrinolysis. The plasma level of plasmin inhibitor was also measured. Neutrophil-activating peptide 2 (NAP-2) was measured as an indicator of platelet 
-granule release. Formation of complexes between platelets and monocytes were quantified in flow cytometry and served as an additional marker of platelet activation. Blood samples for analysis of F1 + 2, TAT, plasmin inhibitor, and NAP-2 were drawn and immediately centrifuged. The plasma samples were frozen immediately after centrifugation and kept at –70°C until they were analyzed in batch. The concentration of F1 + 2 and TAT was measured in citrate plasma using enzyme immunoassay kits (Dade-Behring, Marburg, Germany). The plasma activity of plasmin inhibitor was measured in citrate plasma by a chromogenic substrate assay (Chromogenix Instrumentation Laboratory Company, Lexington MA). NAP-2 concentrations were analyzed in citrate theophylline adenosine dipyridamol plasma using an enzyme immunoassay (NAP-2 duoset, R&D Systems, Abingdon, UK). At each sampling point 50 µL blood was immediately fixed in 200 µL Tyrode's buffer containing 0.35% human serum albumin and 250 µL 1% paraformaldehyde and kept at 4°C until parallel staining for flow cytometry of all 4 samples from each patient. The samples were stained with a PE-conjugated anti-CD41 antibody (GP IIb, Dako, Glostrup, Denmark), which binds strongly to platelets. A negative control antibody (Dako) was also used. Platelet conjugates with monocytes were then identified as CD41-positive cells in the scatter regions of monocytes on a forward scatter/side scatter plot, using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Division of the number of aggregates by the total number of monocytes within the region and multiplication by 100 yielded the stated percentages of platelet-monocyte aggregates.
The lower limit of measurement range for the hemostatic variables were as follows: platelet counts 5 x 109/L, INR 0.8, APTT 15 s, fibrinogen 0.1 g/L, d-dimer 0.2 mg/L, antithrombin 15%, F1 + 2 0.04 nmol/L, TAT 2 µg/L, plasmin inhibitor 5%, and NAP-2 15 pg/mL.
In addition to the primary study outcome measures, heparin and protamine doses, ACT values, postoperative blood loss volumes, amount of postoperative blood autotransfused to the patient, and other transfusion requirements were also recorded. The time from termination of CPB to skin closure was recorded as a measure for the time spent on surgical hemostasis during the final part of the operation.
We hypothesized that possible differences in coagulation variables between elderly and younger cardiac surgery patients would be of the same magnitude as the differences found in the noncardiac surgical population studied by Boldt et al. (7). Boldt et al.'s data were therefore used to calculate the required number of individuals per group aiming for a power of 0.8 and an of 0.05.
Data are presented as mean (sd) and median (range). Statistical analyses were performed using SPSS for Windows®, version 12.0 (SPSS Inc., Chicago, IL). Baseline patient data were compared using Student's t-test or Wilcoxon-Mann-Whitney U-test for scale variables and Fisher's exact test for categorical variables. If necessary, variables were logarithmically transformed to show an acceptable fit to the normal distribution. Repeated measurement analysis of variance was used for analysis of variables measured more than once, using the log-transformed data. This method simultaneously tests for a constant difference between the groups, a change in the measured variable by time, and whether there is an interaction indicating a different change by time in the two groups. When repeated measurement analysis of variance showed a statistically significant interaction and the interaction was considered to be of clinical interest, the difference between the preoperative and the specific postoperative values was calculated. The calculated difference was then compared using Student's t-test. P values < 0.05 were considered statistically significant.
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Results |
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The total doses of heparin and protamine given and the ACT values before and after the administration of heparin and after the administration of protamine, are presented in Table 2. There were no differences between the groups.
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Postoperative bleeding volumes, volume of shed blood autotransfused, time from termination of CPB to skin closure, and packed red blood cell transfusion requirements for the whole hospital stay are presented in Table 3. Postoperative bleeding the first 4 h after surgery and the total number of packed red blood cells given was larger in the elderly patients. Three elderly patients received transfusion of packed red blood cells during the first 20 h postoperatively, the time period during which the postoperative blood samples were drawn. None of the younger patients received packed red blood cells during the first 20 h postoperatively. One elderly patient received 2 units of fresh-frozen plasma 3 h postoperatively because of increased bleeding that was considered to be of nonsurgical origin. No patients received platelet transfusions. One elderly patient was reexplored 2 h postoperatively because of surgical bleeding.
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Antithrombin, F1 + 2, and TAT measurements are shown in Figure 1. The elderly patients had significantly smaller antithrombin concentrations compared with the younger group (P < 0.001). F1 + 2 and TAT concentrations were largest in the elderly patients (F1 + 2 P = 0.004, TAT P = 0.047).
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d-dimer and plasmin inhibitor concentrations are presented in Figure 2. The levels of both variables were highest in the elderly patients (d-dimer P < 0.001, plasmin inhibitor P = 0.003). For d-dimer there was also a significant interaction between the groups (P = 0.022). The difference between concentrations measured 3 h after surgery and preoperative values was larger in the elderly patients compared with the younger patients (17.8 [7.8] mg/L versus 11.1 [7.2] mg/L; P = 0.003).
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Platelet counts, NAP-2 measurements, and flow cytometry results are presented in Figure 3. Platelet counts were smaller in the elderly patients (P = 0.001), whereas NAP-2 was larger in the elderly (P = 0.011). For NAP-2 there was also a significant interaction between the groups (P < 0.001). The difference between values measured 3 h after surgery and preoperative values was larger in the elderly patients compared with the younger patients (49.2 [27.6] ng/mL versus 28.0 [15.8] ng/mL; P = 0.002). Flow cytometry revealed no difference in the percentages of platelet-monocyte complexes between the groups (P = 0.14).
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There were no differences between the elderly and the younger patients in APTT (P = 0.54) or fibrinogen (P = 0.11). For INR there was a small but statistically significant difference between the 2 groups (P = 0.016). This difference was considered not to be of clinical importance as the maximum mean INR in any group at any time point was 1.3.
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Discussion |
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Despite the relatively large doses of heparin given to CABG patients, thrombin and plasmin are generated progressively during CPB (9–11). Large concentrations of TAT and F1 + 2 suggesting coagulation activation have been shown consistently in the literature (9,10,12,13), consistent with our results. Thrombin production plays an important role in the development of hemostatic dysfunction. Excessive thrombin generation may result in consumption of both platelets and coagulation factors, platelet dysfunction, and in excessive fibrinolysis (11,12). The hyperfibrinolysis is to a large extent caused by the release of tissue plasminogen activator during and after CPB that is mediated by activation of coagulation (13–15). Excessive thrombin generation may therefore lead to hemostatic dysfunction with increased postoperative bleeding (11,12,14). As we have shown that elderly patients had increased activation of the hemostatic system indicating increased thrombin generation during CABG compared with younger patients, our results may explain the increased risk for excessive postoperative bleeding seen in elderly cardiac surgery patients.
Marked activation of fibrinolysis is a typical finding in publications investigating the effects of CPB (10,13,14). We also demonstrated considerable fibrinolytic activity causing an increase in d-dimer. The antifibrinolytic drugs aprotinin, tranexamic acid, and -aminocaproic acid have all been shown to reduce bleeding after cardiac surgery (16,17). As elderly patients had more extensive fibrinolysis postoperatively compared with younger patients, it is possible that prophylactic use of antifibrinolytic drugs can also reduce bleeding after cardiac surgery in this group of patients. Further prospective and randomized studies should be performed to determine whether antifibrinolytic therapy can reduce postoperative bleeding in elderly patients undergoing cardiac surgery.
There is no universal method for measuring platelet activation. Quantification of mediators formed during -granule release is one possibility, and, in the present study, measurement of NAP-2 showed that elderly patients had increased platelet activation compared with younger patients (Fig. 3). During platelet activation, up-regulation of adhesions molecules results in formation of platelet-leukocyte complexes. We have previously shown that platelet-monocyte aggregates are formed during cardiac surgery (18), demonstrating platelet activation at hospital discharge. In the present study, the percentage of platelet-monocyte complexes increased markedly after surgery, but there were no differences between elderly and younger patients. Numerous studies using a variety of methods have shown that CPB leads to platelet activation. This may result in adherence of platelets to the surface of the extracorporeal circuit while less active platelets remain in the circulation, contributing to the platelet function defect seen after CPB (19). By flow cytometry, only circulating platelets may be evaluated, and the most activated platelets that are adherent to the extracorporeal circuit or the patient's microvasculature are inaccessible. This may explain why the NAP-2 measurements were more sensitive at detecting the inter-group differences, as released granule products may still reach the circulation.
Table 1 shows that the elderly patients were smaller compared with the younger patients, and the elderly patients probably had a smaller blood volume. It is therefore possible that the elderly patients were hemodiluted to a larger extent by the Ringer's acetate solution in the CPB priming compared with the younger patients. Hemodilution may have influenced the study results to some degree. The effect of hemodilution would, however, be expected to diminish with time, when the priming fluid is redistributed extravascularly. Therefore, a possible effect of hemodilution would be largest for the sampling point 30 minutes after CPB, whereas an effect of hemodilution would be quite small 20 hours after surgery. Also, hemodilution would not affect the preoperative measurements. Repeated measurements analysis of variance show a constant intergroup difference in hemostatic variables through the entire study period, and these differences cannot be explained by hemodilution alone. However, hemodilution may have influenced the plasma concentrations measured 30 minutes and 3 hours after surgery, resulting in the measurement of falsely small concentrations in the group of elderly patients. This would only have affected the difference between groups in antithrombin and platelet counts, variables with decreased levels in the group of elderly patients compared with the control group (Figs. 1 and 3). For the variables F1 + 2, TAT, d-dimer, plasmin inhibitor, and NAP-2, the levels measured were increased in the elderly patients compared with the control patients through the study period despite the possibility of having measured falsely small concentrations in the elderly patients (Figs. 1, 2, and 3). We therefore conclude that a possible effect of hemodilution did not affect the main results and conclusions of the study.
There was a statistically significant difference in postoperative bleeding between the elderly and the younger patients over the first 4 hours after surgery, and the elderly patients were transfused with more units of packed red blood cells (Table 3). As shown in Figures 1, 2 and 3, the activation of platelets, coagulation, and fibrinolysis was most pronounced the first hours after surgery, returning towards preoperative levels after 20 hours. Elderly patients had greatest activation. In particular, the postoperative increase in platelet and fibrinolytic activation was relatively larger in the elderly patients compared with the younger patients, and this may explain the increased postoperative bleeding seen in the elderly the first hours after surgery. An association between variables of activation of the hemostatic system and excessive blood loss after coronary surgery has also been shown previously (20). Moreover, elderly patients had smaller preoperative hemoglobin concentrations compared with the younger patients (Table 1), which, together with the increased early bleeding, explains the larger number of packed red blood cell transfusions given. We did not find a difference in total postoperative bleeding between the two groups. One explanation may be that the study was not powered to identify differences in postoperative bleeding. Also, the total bleeding volumes in the present study were quite small compared with the bleeding volumes reported after CABG in previous investigations (21). The small total bleeding volumes may also be an explanation of the lack of difference in total postoperative bleeding between the two groups.
The possibility that differences in transfusions may have influenced the study results must be considered. No patients in the control group received any transfusions of blood products during the study period. However, 3 elderly patients received transfusions of packed red blood cells before the last blood samples were drawn 20 hours after surgery, whereas 1 elderly patient received 2 units of fresh-frozen plasma. The exclusion of these four patients from the analyses did not change any of the study results, and we conclude that the transfusions of blood products did not influence the main findings of the investigation.
We have shown that compared with a group of younger patients, elderly patients had increased activation of the coagulation system during the perioperative phase of coronary surgery. This may lead to consumption of platelets and coagulation factors, resulting in increased postoperative bleeding. In particular, the elderly patients had more extensive fibrinolysis postoperatively. Further prospective studies are needed to determine whether antifibrinolytic treatment can reduce bleeding after cardiac surgery in elderly patients.
The authors thank Anne Hole for performing the F1 + 2 and TAT analyses, Kari Bratberg for performing the plasmin inhibitor analyses, Toril Holien for performing the NAP-2 analyses, and Hilde Eikemo, Toril Anita Weisethaunet, Marit Aarhaug, and Toril Holien for performing flow cytometry.
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Footnotes |
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Supported, in part, by The Norwegian Health Association, Grant 6432 to Dr. Pleym and by The Research Foundation at St. Olav University Hospital and the Alf and Aagot Helgesens Legacy.
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  H. Pleym, V. Videm, A. Wahba, A. Asberg, T. Amundsen, L. Bjella, O. Dale, and R. Stenseth Heparin resistance and increased platelet activation in coronary surgery patients treated with enoxaparin preoperatively. Eur. J. Cardiothorac. Surg., June 1, 2006; 29(6): 933 - 940. [Abstract] [Full Text] [PDF]
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