HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Eivind Øvrum Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Flom-Halvorsen, H. I. Articles by Brosstad, F. Search for Related Content PubMed PubMed Citation Articles by Flom-Halvorsen, H. I. Articles by Brosstad, F. Related Collections Extracorporeal circulation

Ann Thorac Surg 2003;76:744-748
© 2003 The Society of Thoracic Surgeons

---

Original article: cardiovascular

Quality of intraoperative autologous blood withdrawal used for retransfusion after cardiopulmonary bypass

^a Oslo Heart Center, Research Institute for Internal Medicine, University of Oslo, Oslo, Norway
^b Rikshospitalet, Oslo, Norway

Accepted for publication February 14, 2003.

^* Address reprint requests to Flom-Halvorsen, PhD, Research Institute for Internal Medicine, Sognsvannsveien 20, The National Hospital, N-0027, Oslo, Norway
e-mail: hannef{at}klinmed.uio.no

	Abstract

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
BACKGROUND: Intraoperative autologous blood withdrawal protects the pooled blood from the deleterious effects of cardiopulmonary bypass. Following reinfusion after cardiopulmonary bypass, the fresh autologous blood contributes to less coagulation abnormalities and reduces postoperative bleeding and the need for allogeneic blood products. However, few data have been available concerning the quality and potential activation of fresh blood stored at room temperature in the operating room.

METHODS: Forty coronary artery bypass grafting patients undergoing a consistent intraoperative and postoperative autotransfusion protocol had a median of 1,000 mL of autologous blood withdrawn before cardiopulmonary bypass. After heparinization the blood was drained from the venous catheter via venous cannula into standard blood bags and stored in the operating room until termination of cardiopulmonary bypass. Samples for hemostatic and inflammatory markers were taken from the pooled blood immediately before it was returned to the patient.

RESULTS: There was some activation of platelets in the stored autologous blood, as measured by an increase of ß-thromboglobulin. Indications of thrombin formation, as assessed by plasma levels of thrombin-antithrombin complex and prothrombin fragment 1.2 were not seen, and there was no fibrinolytic activity. The red blood cells remained intact, indicated by the absence of plasma free hemoglobin. As for the inflammatory response, the levels of the terminal complement complex remained stable, and the cytokines tumor necrosis factor- and interleukin 6 levels were not increased during storage. The complement activation products increased minimally, but remained within normal ranges.

CONCLUSIONS: Except for slight activation of platelets, there was no indication of coagulation, hemolysis, fibrinolysis, or immunologic activity in the autologous blood after approximately 1 hour of operating room storage. The autologous blood was preserved in a condition of high quality, and retransfusion after cardiopulmonary bypass represents an uncomplicated and almost costless procedure for blood conservation.

	Introduction

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Intraoperative withdrawal of autologous blood before cardiopulmonary bypass (CPB) is suggested to protect the blood components from degradation and destruction caused by the blood contact with the artificial surfaces of the extracorporeal circuit. Retransfusion of fresh autologous blood has been documented to increase platelet counts and fibrinogen in the postbypass period [1–4] and reduce postoperative bleeding and subsequently the requirement for allogeneic blood transfusions [2, 5]. An additional effect is that the acute intraoperative hemodilution after blood withdrawal reduces hemoglobin loss during the operation.

For best hemostatic effect the heparinized blood is stored at room temperature during the operation and returned to the patient after termination of CPB. Little is known, however, about potential activation of the autologous blood during storage and the quality of the blood before retransfusion. The few studies performed on the autologous blood have only focused on platelet counts and function, and have yielded inconsistent results [3, 6]. The aim of this study was to investigate coagulation, hemolysis, fibrinolysis, platelets, and the immunologic system of pooled blood from patients undergoing coronary artery bypass grfting surgery with CPB.

	Material and methods

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Patients
Forty male patients admitted for first-time, elective coronary artery bypass grafting were randomly selected to the study. Exclusion criteria were patients on anticoagulation, patients receiving antiplatelet therapy less than 7 days preoperatively, and patients with unstable angina. Also patients with an estimated hematocrit less than 22% during CPB were excluded owing to substantial blood sample removal. Left ventricular impairment or left main coronary artery stenosis was not considered as an exclusion criterion. When eligible, the study patients were selected during a 5-month period. The regional ethics committee approved the study protocol, and informed consent was obtained from all patients.

Operative procedures
After heparinization and insertion of the cannulas for CPB, autologous blood was drained from the venous catheter via venous cannula by gravity, immediately before the initiation of bypass, into 1 to 2 blood bags (Baxter Healthcare, Irvine, CA) and stored on the countertop at room temperature. To prevent hemodynamic instability, the blood volume was replaced with priming solution (Ringer’s acetate) through the aortic cannula. The pooled blood did not contain any priming solution or addition of extra heparin or other anticoagulants. After termination of CPB, the autologous blood was returned to the patient before leaving the operating room. The storage time of the autologous blood equals the CPB time. This procedure was an integrated part of the institutional blood conservation protocol [7], which includes the use of cardiotomy suction during the operation and returning of all contents of the extracorporeal circuit to the patient, as well as autotransfusion of the shed mediastinal blood until 18 hours after the operation. There was no increased need for protamine after transfusion of heparinized blood, and no antifibrinolytic drug or other agents to reduce bleeding were routinely given.

The volume of autologous blood removal was guided by an estimated hemodilution during CPB to be more than 0.22 and was calculated by the following formula:

where EBV is estimated blood volume, ABW is autologous blood volume to be withdrawn, RV is replacement volume, HCT is preoperative hematocrit, PV is prime volume, and CV is cardioplegia volume.

Blood sampling
Blood samples from the patients were harvested before the operation and drawn with a syringe from the central venous cannula, discarding the first 10 mL. The bag with autologous blood was turned end-over-end three times for complete mixing of plasma and blood, and samples were drawn with a syringe by means of a three-way stopcock immediately before retransfusion.

Blood tests
Blood specimens from the autologous blood and from the patients were injected into either (1) EDTA-K3 Vacutainers (15%, 0.34 M; Becton Dickinson & Co, Rutherford, NJ) for complement/cytokine analysis and platelet count, (2) sodium-citrate Vacutainers (1/10 volume of 0.129 mol/L; Becton Dickinson) for coagulation and fibrinolysis analysis, or (3) DiatubeH Vacutainers (Diagnostica Stago, Asnieres-sur-Seine, France) for ß-thromboglobulin (ß-TG) determination. All specimens, except for those for whole-blood platelet count, were immediately cooled on ice slush and centrifuged as soon as possible: EDTA Vacutainers: 1,500 g, 10 minutes, 4°C; sodium-citrate Vacutainers: 1,500 g, 10 minutes, room temperature; DiatubeH Vacutainers: 2,500 g, 30 minutes, 4°C. Plasma samples were stored at -70°C until assayed in batches.

Analyses
The thrombin-antithrombin (TAT) complex, the prothrombin fragment 1.2 (PF1.2), and the plasmin/₂-antiplasmin (PAP) complex were all assayed using an enzyme-linked immunosorbent assay according to the instructions given by the manufacturer (Enzygnost TAT/F1 + 2/PAP micro; Behringwerke AG, Diagnostica, Marburg, Germany). The platelet activation marker ß-TG was measured with an enzyme-linked immunosorbent assay as described by the manufacturer (Asserachrom ß-TG; Diagnostica Stago, France). Fibrinogen concentration was determined according to the method of Clauss [8]. For measurement of the heparin cofactor activity of antithrombin (AT), a chromogenic assay was used in accordance with the manufacturer’s instructions (Coamatic Antithrombin; Chromogenix AB, Mölndal, Sweden). The concentration of plasma free hemoglobin (pHgb) was measured by a colorimetric method according to Hunter and associates [9] modified by us for use on Titertek Twinreader Plus (Labsystems Oy, Finland). Platelet count was determined using an automatic cell counter (Cobas Minos ST; Roche, Basel, Switzerland).

The concentration of the fluid-phase terminal complement complex (TCC) was measured according to the method of Mollnes and colleagues [10], which is based on a double antibody enzyme-linked immunosorbent assay with monoclonal antibodies specific for a neoantigen of polymeric C9. The C3 activation product (C3bc) was quantified in a double antibody enzyme-linked immunosorbent assay described by Garred and coworkers [11], using monoclonal antibodies specific for the C3 neoepitope expressed on C3b, iC3b, and C3c as capture antibody. An immunoenzymometric assay (EASIA; Enzyme Amplified Sensitivity Immunoassay) was used for the quantitative measurement of the cytokine tumor necrosis factor- (TNF-) and the cytokine interleukin 6 (IL-6) in accordance with the manufacturer’s instructions (Medgenix Diagnostics, Fleurus, Belgium). The activated clotting time (ACT) was analyzed with High Range HemoTec Automated Coagulation Timer (Medtronic, Inc, Denver, CO).

Statistics
Data are presented as median with ranges. Longitudinal changes between two time points only were analyzed using the paired Student’s t test. A p value less than 0.05 was considered significant for the statistical tests. All data were recorded prospectively and stored in a database.

	Results

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Patient demographics are given in Table 1. All patients had autologous blood withdrawn before CPB in a median amount of 1,000 mL (range, 350–1,650 mL; Table 2). The lowest median hematocrit during CPB was 23% (range, 19%–27%). Postoperative bleeding was moderate, and no bank blood transfusions were given to this selected group of patients. The clinical course was uneventful in all patients (Table 3).

Storage of autologous blood in blood bags did not lead to indications of thrombin formation, as assessed by the plasma levels of TAT complex and PF 1.2, and no fibrinolytic activity was detected (Table 4). Despite numeric statistically significant differences between the autologous withdrawal blood and the preoperative levels in the patients, the values remained within normal ranges.

Concerning the inflammatory response, neither the TCC nor the cytokines tumor necrosis factor- and interleukin 6 increased in the pooled blood during storage (Table 4). The median complement activation product C3bc was increased by 1 AU/mL compared with preoperative level, but was still within the normal range of 7.5 to 11.0 AU/mL (Table 4).

No statistical correlation was found between the time of storage and the laboratory results. Potential effects on the patients’ circulating blood after retransfusion could not be evaluated, as several autotransfusion procedures were performed simultaneously.

	Comment

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
As a result of exposure to large nonendothelial surfaces of the heart-lung machine, the coagulation and fibrinolytic system, as well as blood cells, particularly the platelets, are highly activated [12]. Also markers of the inflammatory response are elevated during CPB [12, 13]. To save some of the patient’s blood from these deleterious effects, predonation before CPB is a logical measure to improve hemostasis after the operation. Provided the patient has a normal preoperative hemoglobin concentration, the procedure of autologous blood removal immediately before CPB is easy to implement and carries only the costs of a standard plastic blood bag. The technique is combined with isovolumetric substitution from the priming volume of the heart-lung machine, which prevent circulatory instability. In our institution, this procedure has proved to be safe and uncomplicated in more than 6,500 consecutive coronary artery bypass grafting patients.

The present data document that the pooled blood is well preserved after approximately 1 hour of storage at room temperature. Apart from a slight elevation of ß-TG, no indications of hemolysis, coagulation, fibrinolysis, complement activation, or increase of cytokines were recorded. Several other techniques for collecting blood intraoperatively have been reported [14, 15]. In one report [16] storage of fresh whole blood in a refrigerator (4°C) was less beneficial toward hemostatic effects compared with storage at room temperature. Withdrawal of heparinized blood into preservative-free bags from an indwelling venous line was found superior with regard to effects on platelet counts and partial thromboplastin times when compared with nonheparinized blood withdrawn into citrated bags [14].

The preservation of platelets is particularly important in the stored autologous blood. Other authors have demonstrated an improvement in clot quality in patients after reinfusion of autologous blood [17]. In addition to platelet counts, platelet adhesiveness has been shown to be preserved during storage [3]. Compared with standard apheresis and whole-blood platelet concentrates, autologous platelets are found to be significantly less activated and more responsive [18].

Expression of the platelet membrane phosphatidylserine is an important cofactor in the generation of thrombin from prothrombin. Preparation of platelet concentrates from platelet-rich plasma is associated with increased platelet-dependent thrombin-generating capacity [19]. In our study, there were no indications of thrombin formation in the stored autologous blood as assessed by the plasma levels of TAT complex and PF1.2. From a practical and clinical perspective, it is noteworthy that the functional impact of platelets in 1 U of fresh whole blood reinfused after CPB is equivalent to that of 10 U of platelet concentrates [20].

As for the timing of retransfusion, studies have demonstrated that the optimum benefit from platelets is obtained when retranfusion is performed within 6 hours after withdrawal [21]. In our practice, the autologous blood is returned to the patient immediately after the end of CPB, with minimal time of storage.

We have previously shown a marked elevation of markers for immunologic activation in the mediastinal shed blood [12]. In the stored autologous blood, however, no activation of the complement system or increase of cytokines was recorded, underscoring the benign nature of the pooled blood. It should be remembered that in bank blood or blood products, both cytokines and/or factors of the complement cascade are found to be activated in whole blood and plasma [22–24], platelet concentrates [25], and red blood cells [24].

In conclusion, the present study clearly indicates that the autologous blood withdrawn before CPB and stored in the operating room represents an ideal blood product to restore hemostatic effects and hemoglobin concentration after CPB. Intraoperative hemodilution even enhanced the blood-saving effects by reducing the intraoperative hemoglobin loss. The costs are negligible, and the procedure is easy to implement in routine practice. The effect on the patients was not investigated in this study, as the autologous withdrawal procedure was an integrated part of a blood conservation protocol including the use of intraoperative cardiotomy suction and autotransfusion of mediastinal shed blood postoperatively.

	Acknowledgments

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
We thank Tom Eirik Mollnes, MD, PhD, for performing the complement analyses.

	References

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 

1. Iyer V.S., Russell W.J. Fresh autologous blood transfusion and platelet counts after cardiopulmonary bypass surgery. Anaesth Intensive Care 1982;10:348-352.[Medline]
2. Schönberger J.P.A.M., Bredèe J.J., Tjian D., Everts P.A.M., Wildevuur C.R.H. Intraoperative predonation contributes to blood saving. Ann Thorac Surg 1993;56:893-898.[Abstract]
3. Hardesty R.L., Bayer W.L., Bahnson H.T. A technique for the use of autologous fresh blood during open-heart surgery. J Thorac Cardiovasc Surg 1968;56:683-688.[Medline]
4. Ochsner J.L., Mills N.L., Leonard G.L., Lawson N. Fresh autologous blood transfusions with extracorporeal circulation. Ann Surg 1973;177:811-817.[Medline]
5. Kochamba G.S., Pfeffer T.A., Sintek C.F., Khonsari S. Intraoperative autotransfusion reduces blood loss after cardiopulmonary bypass. Ann Thorac Surg 1996;61:900-903.[Abstract/Free Full Text]
6. Harris SN, Rinder C, Bonan J, Hines RL. Evaluation of platelet function during autologous blood donation [ASA Abstract A1121]. Anesthesiology 1991;75:No3A
7. Øvrum E., m Holen E., Abdelnoor M., Øystese R. Conventional blood conservation techniques in 500 consecutive artery bypass operations. Ann Thorac Surg 1991;52:500-505.[Abstract]
8. Clauss A. Gerinnungsphysiologische Schnellmetode zur Bestimmung des Fibrinogens. Acta Haematol 1957;17:237-246.[Medline]
9. Hunter F.T., Grove-Rasmussen M., Soutter L. A spectrophotometric method for quantitating hemoglobin in plasma or serum. Am J Clin Pathol 1950;20:429-433.
10. Mollnes T.E., Lea T., Frøland S.S., Harboe M. Quantification of the terminal complement complex in human plasma by an enzyme-linked immunosorbent assay based on monoclonal antibodies against a neoantigen of the complex. Scan J Immunol 1985;22:197-202.[Medline]
11. Garred P., Mollnes T.E., Lea T. Quantification in enzyme-linked immunosorbent assay of a C3 neoepitope expressed on activated human complement factor C3. Scand J Immunol 1988;27:329-335.[Medline]
12. Flom-Halvorsen H.I., Øvrum E., Tangen G., Brosstad F., Ringdal M.A.L., Øystese R. Autotransfusion in coronary artery bypass grafting: disparity in laboratory tests and clinical performance. J Thorac Cardiovasc Surg 1999;118:610-617.[Abstract/Free Full Text]
13. Baksaas S.T., Flom-Halvorsen H.I., Ovrum E., Videm V., Mollnes T.E. Leucocyte filtration during cardiopulmonary reperfusion in coronary artery bypass surgery. Perfusion 1999;14:107-117.[Abstract/Free Full Text]
14. Kaplan J.A., Cannarella C., Jones E.I., Kutner M.H., Hatcher C.R., Jr, Dunbar R.W. Autologous blood transfusion during cardiac surgery. A re-evaluation of three methods. J Thorac Cardiovasc Surg 1977;74:4-10.[Abstract]
15. Cross M.H. Autotransfusion in cardiac surgery. Perfusion 2001;16:391-400.[Abstract/Free Full Text]
16. Golan M., Modan M., Lavee J., et al. Transfusion of fresh whole blood stored (4°C) for short period fails to improve platelet aggregation on extracellular matrix and clinical hemostasis after cardiopulmonary bypass. J Thorac Cardiovasc Surg 1990;99:354-360.[Abstract]
17. Whitten C.W., Allison P.M., Latson T.W., et al. Evaluation of laboratory coagulation and lytic parameters resulting from autologous whole blood transfusion during primary aortocoronary artery bypass grafting. J Clin Anesth 1996;8:229-235.[Medline]
18. Crowther M., Ford I., Jeffrey R.R., Urbaniak S.J., Greaves M. Quality of harvested autologous platelets compared with stored donor platelets for use after cardiopulmonary bypass procedures. Br J Haematol 2000;111:175-181.[Medline]
19. Shapira S., Friedman Z., Shapiro H., Presseizen K., Radnay J., Ellis M.H. The effect of closer storage on the expression of platelet membrane phosphatidylserine and the subsequent impact on the coagulant function of stored platelets. Transfusion 2000;40:1257-1263.[Medline]
20. Mohr R., Martinowitz U., Lavee J., Amroch D., Ramot B., Goor D.A. The hemostatic effect of transfusing fresh whole blood versus platelet concentrates after cardiac operations. J Thorac Cardiovasc Surg 1988;96:530-534.[Abstract]
21. Levin R.H., Freirich E.J., Chappel W. Effect of storage up to 48 hours on response to transfusion of platelet rich plasma. Transfusion 1964;4:251-256.[Medline]
22. Hyllner M., Arnestad J.P., Bengtson J.P., Rydberg L., Bengtsson A. Complement activation during storage of whole blood, red cells, plasma and buffy coat. Transfusion 1997;37:264-268.[Medline]
23. Schleuning M., Schmid-Haslbeck M., Utz H., et al. Complement activation during storage of blood under normal blood bank conditions. Effects of proteinase inhibitors and leukocyte depletion. Blood 1992;79:3071-3075.[Abstract/Free Full Text]
24. Weisbach V., Wanke C., Zingsem J., Zimmermann R., Eckstein R. Cytokine generation in whole blood, leukocyte-depleted and temporarily warmed red blood cell concentrates. Vox Sang 1999;76:100-106.[Medline]
25. Hetland G., Mollnes T.E., Bergh K., Hogasen K., Bergerud U.E., Solheim B.G. Effect of filtration and storage of platelet concentrates on the production of the chemotaxins C5a, interleukin 8, tumor necrosis factor alpha, and leukotriene B4. Transfusion 1998;38:16-23.[Medline]

This article has been cited by other articles:

				S. Cromheecke, S. Lorsomradee, P. J. Van der Linden, and S. G. De Hert Moderate Acute Isovolemic Hemodilution Alters Myocardial Function in Patients with Coronary Artery Disease Anesth. Analg., October 1, 2008; 107(4): 1145 - 1152. [Abstract] [Full Text] [PDF]

				M. Westerberg, J. Gabel, A. Bengtsson, J. Sellgren, O. Eidem, and A. Jeppsson Hemodynamic effects of cardiotomy suction blood J. Thorac. Cardiovasc. Surg., June 1, 2006; 131(6): 1352 - 1357. [Abstract] [Full Text] [PDF]

				J. Lavee Invited commentary Ann. Thorac. Surg., September 1, 2003; 76(3): 748 - 748. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Eivind Øvrum Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Flom-Halvorsen, H. I. Articles by Brosstad, F. Search for Related Content PubMed PubMed Citation Articles by Flom-Halvorsen, H. I. Articles by Brosstad, F. Related Collections Extracorporeal circulation