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Ann Thorac Surg 2001;72:1598-1602
© 2001 The Society of Thoracic Surgeons

Original article: cardiovascular

Survival, function, and hemolysis of shed red blood cells processed as nonwashed blood and washed red blood cells1

C. Robert Valeri, MD*a, Richard C. Dennis, MDa, Gina Ragnoa, Linda E. Pivacek, MPHa, Herbert B. Hechtman, MDa, Shukri F. Khuri, MDb,c

a Naval Blood Research Laboratory and Surgical Service, Boston University School of Medicine, Boston, Massachusetts, USA
b Surgical Service, Veterans Affairs Boston Healthcare System, West Roxbury, Massachusetts, USA
c Harvard Medical School, Boston, Massachusetts, USA

Accepted for publication July 16, 2001.

* Address reprint requests to Dr Valeri, Naval Blood Research Laboratory, Boston University School of Medicine, 615 Albany St, Boston, MA 02118, USA
e-mail: navblood{at}bu.edu


   Abstract
Top
 Footnotes
 Abstract
Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Background. Shed nonwashed blood and shed washed red blood cells (RBC) are being used as alternatives to allogeneic liquid-preserved RBC for patients during thoracic and cardiovascular surgical procedures.

Methods. Mongrel dogs were bled a volume of blood into the abdominal cavity and the shed blood was reinfused as nonwashed blood or washed RBC. The 51Cr RBC volumes were measured before, immediately after, and 24 hours after the exchange transfusion to assess the recovery of the shed RBC and the 24-hour posttransfusion survival. Compatible dogs were given allogeneic transfusions of 51Cr-labeled nonwashed blood and washed RBC, and 24-hour posttransfusion survival and half-life were measured.

Results. Immediately after the 100% exchange transfusion, the recovery value was 62% for the nonwashed shed blood and 82% for the washed RBC. Both the nonwashed blood and the washed RBC had 24-hour posttransfusion survival values of 90% and normal oxygen transport function after the exchange transfusion. Compatible allogeneic 51Cr-labeled nonwashed blood and washed RBC had normal 24-hour posttranfusion survival and 51Cr half-life values.

Conclusions. The survival, function, and hemolysis of shed nonwashed blood and shed washed RBC were similar to fresh blood in the dog that underwent a 100% exchange transfusion.


   Introduction
Top
 Footnotes
 Abstract
 Introduction
Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Allogeneic transfusions have been associated with disease transmission as well as immunomodulation, characterized postoperatively by increased sepsis [1, 2], increased rates of metastases [3, 4], graft-versus-host disease [5], and immunosuppression [6–8].

Because allogeneic blood carries with it the risk of potentially serious problems, interest has increased in the use of intraoperatively and postoperatively collected shed blood. Although limited amounts of nonwashed shed blood have been safely infused postoperatively to patients undergoing cardiac operations [9], its quality has been questioned, particularly when compared with the reinfusion of washed red blood cells (RBC) [10].

The present study was designed to evaluate a 100% exchange transfusion of the dog’s blood volume to assess the quality and posttransfusion survival of intraoperatively salvaged blood processed as nonwashed blood and washed RBC.


   Material and methods
Top
 Footnotes
 Abstract
 Introduction
 Material and methods
Results
 Comment
 Acknowledgments
 References
 
Twenty-three nonsplenectomized mongrel dogs weighing 18 to 30 kg were studied. The protocol was approved by the Institutional Animal Care and Use Committee at Boston University Medical Center and by the Department of Defense Veterinary Service. All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health publication 85-23, revised 1985), and were housed in conformance with National Institutes of Health guidelines and fed a routine diet.

Twenty-four to 48 hours before the exchange transfusion, the dogs’ RBC and plasma volumes were measured using 2.5 µCi of 51Cr and 0.5 µCi of 125I-labeled albumin as described previously [11]. This measurement was used to determine the volume of blood required to perform the 100% exchange transfusion.

On the morning of the study, each dog was sedated with 2.8 mg/kg of acepromazine intramuscularly. General anesthesia was maintained with intravenous sodium pentobarbital throughout the procedure. A 20-gauge catheter was placed in the femoral artery for withdrawal of blood samples and measurement of mean arterial pressure. A 16-gauge catheter was placed in the femoral vein to administer 0.9% NaCl to maintain the arterial pressure and to reinfuse the shed blood. Each dog was intubated 1 hour before the beginning of the 3-hour procedure and was ventilated mechanically with room air throughout the procedure. The electrocardiogram was monitored throughout the procedure, and a nasal pharyngeal temperature probe was used to monitor and maintain core temperature using warming blankets and heating lamps. A laparotomy was performed and a 16-gauge catheter was placed into the abdominal aorta.

Experimental design
After the laparotomy, 100% of the measured blood volume was removed from the catheterized abdominal aorta. A three-way stopcock with extension tubing was used to withdraw 50-mL aliquots of blood, for a total of 400 to 500 mL. The blood was delivered into the abdominal cavity through the third port of the stopcock. The blood was immediately aspirated and collected into either the Haemonetics Cell Saver Plus washing system (Haemonetics Corporation, Braintree, MA) for processing of the washed RBC or into the Solcotrans collection system (Solco Basle, Inc, Hingham, MA) for processing of the nonwashed blood. The procedure was repeated for collection of each 400- to 500-mL volume of blood. As 100% of the dog’s blood volume was being removed and reinfused, a mean of 1,700 mL of 0.9% sodium chloride was also infused into the dogs.

For both the washed and nonwashed systems, blood was collected and processed according to the manufacturer’s instructions. Briefly, these were as follows:

Haemonetics Cell Saver Plus
Before aspiration of each 400- to 500-mL volume of blood, the suction tubing and reservoir were primed with 100 mL of 0.9% sodium chloride containing 30 U/mL of heparin, with suction maintained between 100 to 200 mm Hg. After aspiration of 200 to 250 mL of blood into the reservoir, the Haemonetics Cell Saver Plus 225 mL disposable bowl was filled to the shoulder at 500 mL/min. If RBC spillage was noticed, the pump speed was reduced.

The RBC were washed with 1,000 mL of 0.9% sodium chloride at 600 mL/min and pumped into the reinfusion bag at 500 mL/min. The washed RBC was returned by gravity through a standard 170-micron blood recipient set and blood warmer coil (Fenwal Laboratories, #4C2403; Baxter Healthcare Corp, Deerfield, IL). The size of the washing bowl limited the volume that could be processed to 200 to 250 mL. While the first 200- to 250-mL volume of washed RBC was being reinfused, the next 200- to 250-mL volume was being washed. The hematocrit of the washed RBC ranged from 30 to 40 V%.

Solcotrans
The pooled blood was aspirated with suction maintained at 80 to 100 mm Hg into the Solcotrans system containing 40 mL of acid-citrate-dextrose (ACD) and was mixed intermittently throughout the process. Each 400- to 500-mL volume of blood was reinfused by gravity through a 40-micron Pall filter (Pall Corporation, East Hills, NY) using a standard recipient set and blood warmer coil. On completion of the reinfusion of the first volume of nonwashed blood, the next 400- to 500-mL volume of blood was collected and processed as described above.

Blood sampling
Arterial blood samples were obtained after the laparotomy was performed and before the first 400- to 500-mL volume of blood was removed for measurements of hemoglobin concentration, hematocrit value, platelet count, plasma hemoglobin, fibrinogen, and factor VIII clotting protein. These measurements were repeated after the reinfusion of each 400 to 500 mL. Red blood cell adenosine triphosphate (ATP), 2,3 diphosphoglycerate (2,3 DPG), and P50 values (partial pressure in mm Hg at which 50% of the RBC were oxygenated) were measured before and after completion of the 100% exchange transfusion. Heparinized arterial blood was obtained to measure pH, pO2, and pCO2 before and after the reinfusion of each 400- to 500-mL volume, and again 24 hours later using the IL 482 Blood Gas Analyzer (Lexington, MA).

Approximately 1 hour after reinfusion of the final 400 to 500 mL of filtered nonwashed blood or washed RBC, the blood volume was measured for the second time using 51Cr-labeled RBC to measure the RBC volume and 125I-labeled albumin to measure the plasma volume [11]. The second RBC volume was measured using autologous blood collected before the exchange transfusion. Ten milliliters of the 51Cr-labeled autologous RBC containing 15 µCi of 51Cr and 5 mL of 125I-labeled albumin containing 1.25 µCi of 125I albumin were infused [11]. Twenty-four hours after the 100% exchange transfusion, the third RBC and plasma volumes were measured using 30 µCi of 51Cr and 5 µCi of 125I albumin. The sequential RBC measurements were used to calculate the 24-hour posttransfusion survival of the washed and nonwashed autologous RBC.

Allogeneic red blood cell survival
Allogeneic RBC survival measurements were performed to confirm the RBC survival measurements obtained using sequential RBC volume measurements in the exchange-transfused dogs.

Twelve healthy splenectomized dogs received compatible allogeneic transfusion of an aliquot from the final 400- to 500-mL volume of nonwashed blood or washed RBC from the exchange transfusion [12]. The dogs’ RBC were typed using antisera to detect dog erythrocyte antigen (DEA) 1.1, DEA 1.1,2, DEA 3, DEA 4, DEA 5, DEA 6, and DEA 7, and were crossmatched using the canine Coombs serum provided by R. Bull, DVM (Michigan State University).

The double 51Cr procedure was used to measure the 24-hour posttransfusion survival and life-span of the compatible allogeneic nonwashed blood and washed RBC as follows: A 10-mL volume of autologous RBC containing 2.5 µCi of 51Cr was used to measure the recipient dog’s RBC volume. A 10-mL aliquot of nonwashed blood or washed RBC from the compatible exchange-transfused dog containing 25 µCi of 51Cr was used to measure the survival by the double 51Cr technique [12, 13]. Blood samples were collected from the recipient dog before and 5, 10, 20, and 30 minutes, 24 and 48 hours, and 7, 14, and 21 days after the allogeneic transfusion for measurements of 24-hour posttransfusion survival and 51Cr half-life values.

Statistical analysis
Data were analyzed using SAS (SAS Institute, Inc, Cary, NC). Means and standard deviations are reported; t tests were used to compare the RBC volume and posttransfusion survival values for the nonwashed and washed groups. For other variables, repeated-measures analysis of variance (ANOVA) was used to test for differences over time within the nonwashed and washed groups. Significant findings were followed by pairwise comparisons. Two-factor analysis of variance was used to test for differences between the two groups over time. Significant interactions of group and time were followed by pairwise comparisons between groups at each time point. All multiple pairwise comparisons were adjusted for experimentwise error using the Bonferroni procedure. A p value of less than 0.05 was considered significant.


   Results
Top
 Footnotes
 Abstract
 Introduction
 Material and methods
 Results
Comment
 Acknowledgments
 References
 
Table 1 reports the 51Cr RBC volumes measured in 23 nonsplenectomized mongrel dogs before, immediately after, and 24 hours after the 100% exchange transfusion of filtered nonwashed blood (n = 14) and filtered washed RBC (n = 9). These data demonstrate that the dogs receiving washed RBC had 82% of their baseline RBC volume immediately after the exchange transfusion, whereas dogs receiving nonwashed blood had 62% of their baseline RBC volume (p < 0.001). In both the washed and nonwashed groups, the RBC volume measured 24 hours after the exchange transfusion was approximately 90% of the immediate postreinfusion value.


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  		Table 1. RBC Volume in Study Dogs

 
Table 2 reports the 24-hour posttransfusion survival values and 51Cr half-life values in 6 dogs that received allogeneic transfusions of nonwashed blood and 6 dogs that received allogeneic transfusions of washed RBC. The 24-hour posttransfusion survival was 91% for nonwashed RBC and 87% for washed RBC, with normal half-life values of 22 days for both groups, indicating similar 24-hour posttransfusion survival values for the allogeneic RBC and those obtained using sequential RBC volume measurements in the exchange-transfused dogs.


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  		Table 2. Twenty-Four Hour Posttransfusion Survival and 51Cr Half-Life of Allogeneic Nonwashed Shed Blood and Washed Shed RBC

 
Table 3 reports normal levels of RBC ATP, DPG, and P50 in the nonwashed blood and the washed RBC after exchange transfusion. There was no significant difference in the mean plasma hemoglobin levels: 26 mg/dL after the 100% exchange transfusion with nonwashed blood, and 14 mg/dL after exchange transfusion with washed RBC (Table 3).


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  		Table 3. Levels of RBC ATP, DPG, P50, and Plasma Hemoglobin in Dogs Before and After Exchange Transfusion

 
The platelet count and fibrinogen levels were significantly reduced at the completion of the exchange transfusion in both the nonwashed and washed groups (Table 4). However, 24 hours after the exchange transfusion, the fibrinogen levels were significantly higher than the baseline levels in both groups. Analysis of variance showed significant interaction between the groups (nonwashed and washed) and time. Pairwise comparisons between groups at each time indicated that the increase in fibrinogen 24 hours after transfusion was greater in the nonwashed group. Arterial pO2 and pCO2 were normal in the two groups (Table 4). Arterial pH was significantly reduced at the completion of the exchange transfusion for both the washed and nonwashed groups. The hematocrit levels and hemoglobin concentrations were significantly higher in the dogs that underwent exchange transfusion with washed RBC than in the dogs receiving nonwashed blood, both at the completion of the exchange transfusion and 24 hours after (Table 4).


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  		Table 4. Various Blood Values Before and After the Exchange Transfusion of Autologous Nonwashed and Washed Shed Blood

 

   Comment
Top
 Footnotes
 Abstract
 Introduction
 Material and methods
 Results
 Comment
Acknowledgments
 References
 
Opinions differ among investigators about the safety and therapeutic benefit of shed blood collected from patients intraoperatively and postoperatively and reinfused as nonwashed blood or as washed RBC [10]. Several investigators have studied the survival of shed nonwashed blood and shed washed RBC in patients undergoing cardiovascular, spinal, aortic, and orthopedic surgical procedures [10, 14–20].

The present study was designed to evaluate the in vivo posttransfusion survival and quality of intraoperatively salvaged blood processed as nonwashed blood and as washed RBC. Shed blood was collected from the dog’s peritoneal cavity and exchange-transfused as nonwashed filtered blood or as washed filtered RBC.

To simulate the operational use of the systems, the blood was collected and processed according to the manufacturer’s instructions. This method resulted in a difference in the anticoagulant solution and negative pressure used during the shed blood collection. Immediately after the exchange transfusion, the dogs receiving nonwashed blood had a significantly reduced RBC volume compared with dogs receiving washed RBC. This difference was related to a reduction in the in vitro recovery of RBC processed as nonwashed blood due to differences in the collection methods.

The quality of intraoperatively collected shed dog blood processed as washed RBC and nonwashed blood was similar to that of fresh dog RBC [12]. The dogs that received allogeneic transfusions of nonwashed blood and washed RBC exhibited 24-hour posttransfusion survival values of about 90%. These data confirmed the results obtained using sequential RBC volume measurements in the exchange-transfused dogs. The 51Cr half-life was not measured in the dogs that underwent the 100% exchange transfusions because the RBC volume in the dogs reinfused with nonwashed blood was reduced by 40% and the RBC volume in dogs reinfused with washed RBC was reduced by 20%. The 51Cr half-life of the RBC could not be accurately measured in these dogs because the RBC volumes were not stable during the 4-week posttransfusion period. Allogeneic nonwashed blood and washed RBC transfusions resulted in normal 51Cr half-life values that were similar to those observed in dog studies previously reported [12]. Measurements of RBC ATP, DPG and P50 demonstrated that both the nonwashed blood and washed RBC had normal oxygen transport function.

At the completion of and 24 hours after the 100% exchange transfusion, hemoglobin concentration and hematocrit values were significantly higher in the dogs receiving washed RBC than in those receiving nonwashed blood. The reinfusion of nonwashed blood and washed RBC resulted in only slightly increased plasma hemoglobin levels (26 and 14 mg/dL, respectively) and normal arterial pO2 and pCO2 values. Arterial pH, factor VIII levels, fibrinogen levels, and platelet counts were significantly reduced in both groups after the exchange transfusion.

Twenty-four hours after the exchange transfusions to both groups, platelet count remained significantly reduced, the factor VIII level returned to normal, and the fibrinogen levels were increased to 200% of baseline. In the dogs with reduced RBC volumes, the increases in the fibrinogen and factor VIII levels reflected the response of the liver and spleen to the 100% exchange transfusion.

Measurements of viability, function, and hemolysis in the shed nonwashed blood and washed RBC for the exchange-transfused dogs were similar to measurements in fresh blood. However, the salvaged shed washed RBC exhibited 20% more viable and functional RBC than the shed nonwashed blood.

The volume of shed nonwashed blood that can be safely infused into patients undergoing thoracic and cardiovascular operations is about 700 mL [9]. For volumes of shed blood greater than 700 mL, we recommend that shed blood should be washed to reduce the potential risks associated with nonwashed blood and to provide a readily available source of viable and functional autologous RBC to reduce the need for preserved allogeneic RBC.


   Acknowledgments
Top
 Footnotes
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
References
 
This work was supported by the U.S. Navy (Office of Naval Research Contracts N00014-79-C-0168, N00014-88-C-0118, N00014-94-C-0149, with funds provided by the Naval Medical Research and Development Command).

The authors acknowledge the editorial assistance of Cynthia A. Valeri and the secretarial assistance of Marilyn Leavy.


   Footnotes
Top
 Footnotes
Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Dr Valeri discloses that he has a financial relationship with Haemonetics Corporation and Solco Basle, Inc.

1 The opinions or assertions contained herein are those of the authors and are not to be construed as official or reflecting the views of the US Navy Department or US Naval Service at large. Back


   References
Top
 Footnotes
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 

  1. Tartter P.I. Blood transfusion and infectious complications after colorectal cancer surgery. Br J Surg 1988;75:789-792.[Medline]
  2. Blumberg N., Heal J.M. Effects of transfusion on immune function. Cancer recurrence and infection. Arch Path Lab Med 1994;118:371-379.
  3. Clarke P.J., Tarin D. Effect of pre-operative blood transfusion on tumour metastases. Br J Surg 1987;74:520-522.[Medline]
  4. Chung M., Steinmetz O.K., Gordon P.H. Perioperative blood transfusion and outcome after resection for colorectal carcinoma. Br J Surg 1993;80:427-432.[Medline]
  5. Anderson K.C., Weinstein H.J. Transfusion-associated graft-versus-host disease. N Engl J Med 1990;323:315-321.[Medline]
  6. Opelz G., Sengar D.P.S., Mickey M.R., et al. Effect of blood transfusion on subsequent kidney transplants. Transplant Proc 1973;5:253-259.[Medline]
  7. Jensen L.S., Kissmeyer-Nielsen P., Wolff B., Qvist N. Randomised comparison of leucocyte-depleted versus buffy-coat-poor blood transfusion and complications after colorectal surgery. Lancet 1996;348:841-845.[Medline]
  8. Bordin J.O., Chiba A.K., Carvalho K.I.L., et al. The effect of unmodified or prestorage white cell-reduced allogeneic red cell transfusions on the immune responsiveness in orthopedic surgery patients. Transfusion 1999;39:718-723.[Medline]
  9. Axford T.C., Dearani J.A., Ragno G., et al. Safety and therapeutic effectiveness of reinfused shed blood after open heart surgery. Ann Thorac Surg 1994;57:615-622.[Abstract/Free Full Text]
  10. In: Tawes R.L., Jr, ed. Autotransfusion. Therapeutic principles and trends. Grosse Pointe, MI: Gregory Appleton, 1997.
  11. Valeri C.R., Donahue K., Feingold H.M., et al. Increase in plasma volume after the transfusion of washed erythrocytes. Surg Gynec Obstet 1986;162:30-36.
  12. Contreras T.J., Lindberg J.R., Lowrie G.B., et al. Liquid and freeze-preservation of dog red blood cells. Transfusion 1979;19:279-292.[Medline]
  13. Szymanski I.O., Valeri C.R. Evaluation of double 51Cr technic. Vox Sang 1968;15:287-292.[Medline]
  14. Ansell J.E., Parrilla N., King M., et al. Survival of auto- transfused red blood cells recovered from the surgical field during cardiovascular operations. J Thorac Cardiovasc Surg 1982;84:387-391.[Abstract]
  15. Ansell J.E., Vander Salm T., Stephenson W., Szymanski I., Fournier L. In vivo survival of red blood cells processed by a bubble or membrane oxygenator during cardiopulmonary bypass surgery. Texas Heart Inst J 1986;13:247-251.
  16. Ray J.M., Flynn J.C., Bierman A.H. Erythrocyte survival after intraoperative autotransfusion in spinal surgery: an in vivo comparative study and 5-year update. Spine 1986;11:879-882.[Medline]
  17. Kent P., Ashley S., Thorley P.J., Shaw A., Parkin A., Kester R.C. 24-hour survival of autotransfused red cells in elective aortic surgery: a comparison of two intra-operative autotransfusion systems. Br J Surg 1991;78:1473-1475.[Medline]
  18. O’Hara P.J., Hertzer N.R., Santilli P.H., Beven E.G. Intraoperative autotransfusion during abdominal aortic reconstruction. Am J Surg 1983;145:215-220.[Medline]
  19. Umlas J., Jacobson M.S., Kevy S.V. Survival and half-life of red cells salvaged after hip and knee replacement surgery. Transfusion 1993;33:591-593.[Medline]
  20. Szymanski I.O., Ansell J.E., Vander Salm T. Effect of blood salvage on red cell survival. In: Tawes R.L., Jr, ed. Autotransfusion. Therapeutic principles and trends. Grosse Pointe, MI: Gregory Appleton, 1997:107-114.




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