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Complement split products and pro-inflammatory cytokines in salvaged blood after hip and knee arthroplasty

From the Department of Anesthesiology & Intensive Care, East Hospital/Sahlgrenska University Hospital, Gothenburg, Sweden.

Address correspondence to: Dr. Iréne Andersson, Department of Anesthesiology & Intensive Care, East Hospital, Sahlgrenska University Hospital, S-416 85 Gothenburg, Sweden. Phone: 46-31-3434000; Fax: 46-31-3434490; E-mail: irene_maria.andersson{at}sahlgrenska.se

	Abstract

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Purpose: To determine whether salvaged autologous blood collected postoperatively contains complement split products (SC5b-9), and pro-inflammatory cytokines (IL-6 and IL-8) and whether there are any differences between blood collected during hip or knee surgery.

Methods: Fifty-eight consecutive patients undergoing hip or knee replacement surgery were studied. Thirty-eight had postoperative bleeding large enough to require infusion of salvaged blood. The salvaged blood was filtered during collection through a 200 µm filter and before infusion a 40 µm filter was used. Samples for complement and cytokine determinations were drawn from the circulation and from the collected blood.

Results: High concentrations of SC5b-9, IL-6, and IL-8 were found in salvaged blood. The concentrations were higher than in the circulation (P < 0.05). The circulating concentrations of IL-6 and IL-8 were increased 60 min and 12-18 hr after transfusion. There were no differences regarding SC5b-9, IL-6, and IL-8 in the blood collected after hip or knee surgery.

Conclusion: Blood collected from a surgical wound contains large concentrations of inflammatory mediators. There were no differences between blood collected during hip or knee surgery.

SEVERAL alternatives to allogeneic blood transfusion have been proposed.^1–3 Different techniques for autologous transfusions have been developed. Autotransfusion can be subdivided depending on the extent to which the scavenged blood is processed prior to infusion.⁴ In filter systems the salvaged blood is anti-coagulated and filtered, whereas in other autotransfusion systems the salvaged blood is washed and centrifuged prior to infusion.

Knee replacement surgery is normally performed in a blood-free field after the application of a tourniquet. The effect on complement activation and cytokine release by the blood-free field is not known. It has been demonstrated previously that collection and infusion of wound blood leads to activation of the coagulation, fibrinolytic and complement systems^5-8 and TNF-, IL-6, IL-8 and PMN elastase are formed during salvage.⁸

The purpose of this study was to determine whether salvaged blood collected postoperatively contains complement split products (SC5b-9), and pro-inflammatory cytokines (IL-6 and IL-8) and whether there are any differences between blood collected during knee or hip surgery.

	Methods

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The Ethics Committee of Sahlgrenska University Hospital, Gothenburg, Sweden approved the study. Fifty-eight consecutive patients undergoing elective hip or knee replacement surgery were studied. Thirty patients underwent hip surgery and 28 knee surgery. Their ages ranged from 36-90 yr (median 72 yr). Spinal anesthesia with was used. Knee replacement surgery was performed in a blood-free field. The tourniquet was applied with a pressure of 100 mmHg above the patients systolic blood pressure. Postoperatively a drainage suction system allowing infusion of aspirated wound blood without using anticoagulation was used (Bellovac® A.B.T, AstraTech, Sweden).

Determinations of SC5b-9, IL-6, IL-8, PMN elastase, hemoglobin, free hemoglobin, hematocrit, leukocytes, platelets, sodium and potassium were made preoperatively, one minute before the start of infusion, and at 60 min and 12-18 hr after completed infusion. Samples were drawn from the infusion bag one minute before start of infusion and eight hours after start of collection of blood. SC5b-9 was determined with a double-antibody enzyme-linked immunosorbent assay. IL-6 and IL-8 were determined with an EIA test (R&D Systems, Abingdon, UK) and PMN elastase was determined by an ELISA method (DPC, Los Angels, USA).

Bacterial cultures were performed from all the infusion bags.

Statistics
Median values and ranges are given. Comparisons were made by repeated ANOVA followed by Wilcoxon signed rank test.

	Results

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There were no differences between the volumes of collected blood obtained during hip and knee surgery. Sixteen of 28 knee surgery patients received a median of 504 mL salvaged blood and 22 of 30 hip surgery patients received a median of 363 mL salvaged blood.

Hemoglobin concentrations in patient blood were lower before the start of infusion, 60 min and 12-18 hr after infusion than the preoperative levels (P < 0.05). Higher concentrations of free hemoglobin were found in the infusion bags than in circulating blood (P < 0.001). There were no differences between hip and knee surgery regarding hemoglobin, hematocrit or free hemoglobin (Tables I, II). The leukocyte count was increased before the start of infusion, 60 min and 12-18 hr after infusion compared with preoperative levels (P < 0.05). The platelets count was decreased in the infusion bag compared with that in circulating blood (P < 0.05).

View larger version (14K): [in this window] [in a new window]   FIGURE 1 IL-6 preoperatively, one minute before start of infusion, 60 min and 12-18 hr after completed infusion in patients undergoing hip surgery (open bars) and patients undergoing knee surgery (filled bars). Median and ranges are given. In hip surgery the median IL-6 concentration in the infusion bag was one minute before start of infusion 4780 pg•mL–1 (range 162-65700 pg•mL–1) and eight hours after start of collection of salvaged blood the median concentration was 4532 pg•mL–1 (range 164-41400 pg•mL–1) In knee surgery the median IL-6 concentration in the infusion bag one minute before start of infusion was 4680 pg•mL–1 (range 252-56000 pg•mL–1) and eight hours after start of collection of salvaged blood the median concentration was 5100 pg•mL–1 (range 218-36600 pg•mL–1).

View larger version (13K): [in this window] [in a new window]   FIGURE 2 IL-8 preoperatively, one minute before start of infusion, 60 min and 12-18 hr after completed infusion in patients undergoing hip surgery (open bars) and patients undergoing knee surgery (filled bars). Median and ranges are given. In hip surgery the median IL-8 concentration in the infusion bag one minute before start of infusion was 1420 pg•mL–1 (range 55-10800 pg•mL–1) and eight hours after the start of collection of salvaged blood the median concentration was 1530 pg•mL–1 (range 82-6330 pg•mL–1) In knee surgery the median IL-8 concentration in the infusion bag one minute before start of infusion was 880 pg•mL–1 (range 90-10400 pg•mL–1) and eight hours after start of collection of salvaged blood the median concentration was 1650 pg•mL–1 (range 44-8580 pg•mL–1).

	Discussion

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This study demonstrated that salvaged blood contains high concentrations of SC5b-9, IL-6, IL-8 and PMN elastase. After infusion of filtered salvaged blood, hypotension, hyperthermia and febrile reactions have been reported.^4,9,10 Febrile non-hemolytic reactions occur in patients receiving platelet transfusions.⁴ In the present study there were no signs of adverse reactions that could be explained by salvaged blood infusion. Salvaged blood also contains high concentrations of free hemoglobin. However, the concentrations of free hemoglobin in the circulation did not increase after infusion of salvaged blood.

The study demonstrated that complement is activated in salvaged blood. Increased plasma concentrations of complement split products, IL-6 and IL-8 may be due to either the infusion itself or the surgical trauma. However, it is not possible to draw any conclusions regarding the origin of the activation. The salvaged blood had been exposed to tissue factors at the operation site, to air, and to synthetic material of the collection equipment. These factors may contribute to the activation of the inflammatory systems.

Filter systems seem to be safe when a low volume of salvaged blood is returned. Filter systems are generally easier and less expensive to handle. In the present study no anticoagulation was used in the infusion system. There were no reported problems with coagulation of the salvaged blood.

Depending on the surgical situation, the hemoglobin and hematocrit in unprocessed salvaged blood are low. If a small volume of blood is infused, there are few side effects. On the other hand, if a large volume of blood is needed, filtered salvaged blood may be dangerous due to activation of the complement cascade and the release of pro-inflammatory cytokines.

This study showed that blood collected from a surgical wound contains large concentrations of inflammatory mediators. There were no differences between blood collected during knee or knee surgery.

	Acknowledgments

 
The study was supported by grants from the Swedish Medical Research council (B2000-17x-11233-01A).

Accepted for publication November 10, 2000.

	References

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3 Williamson KR, Taswell HF. Intraoperative blood salvage: a review. Transfusion 1991; 31: 662–75.[Medline]

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6 Arnestad JP, Bengtsson A, Bengtson JP, Johansson S, Redl H, Schlag G. Release of cytokines, polymorphonuclear elastase and terminal C5b-9 complement complex by infusion of wound drainage blood. Acta Orthop Scand 1995; 66: 329–33.[Medline]

7 Krohn CD, Reikerås O, Mollnes TE, Aasen AO. Complement activation and release of interleukin-6 and tumour necrosis factor- in drained and systemic blood after major orthopaedic surgery. Eur J Surg 1998; 164: 103–8.[Medline]

8 Arnestad JP, Bengtsson A, Bengtson JP, Tylman M, Redl H, Schlag G. Formation of cytokines by retransfusion of shed whole blood. Br J Anaesth 1994; 72: 422–5.[Abstract/Free Full Text]

9 Busund R, Balteskard L, Rönning G, Høgåsen K, Revhaug A. Fatal myocardial depression and circulatory collapse associated with complement activation induced by plasma infusion in severe porcine sepsis. Acta Anesthesiol Scand 1995; 39: 100–8.[Medline]

10 Blevins F T, Shaw B, Valeri C R, Kasser J, Hall J. Reinfusion of shed blood after orthopaedic procedures in children and adolescents. J Bone Joint Surg 1993; 75–A: 363–71.[Abstract/Free Full Text]

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