Modification of Suction-Induced Hemolysis During Cell Salvage

General Article Modification of Suction-Induced Hemolysis During Cell Salvage Jonathan H. Waters, MD* Brandon Williams, BS† Mark H. Yazer, MD, FRCPC‡§ Marina V. Kameneva, PhD†储 BACKGROUND: The efficiency of red blood cell collection during cell salvage is dictated by multiple variables, including suction pressure. In this study, we attempted to determine the influence of suction pressure on the efficiency of cell salvage and to identify methods for minimizing the impact of suction on salvaged blood. METHODS: Whole blood was placed in 60-mL aliquots either in a beaker or on a flat surface and suctioned at 100 and 300 mm Hg. The amount of hemolysis was measured and compared under the varying conditions. The experiments were repeated with the blood diluted with normal saline solution in a 1:1 mix. RESULTS: Hemolysis ranged from 0.21% to 2.29%. Hemolysis was greatest when whole blood was suctioned from a flat surface at 300 mm Hg. It was reduced when the blood was diluted with saline. Blood suctioned from a surgical field during cell salvage should be done with minimal suction pressures and with the goal of minimizing blood–air interfaces. CONCLUSIONS: Significant reduction of blood damage can be obtained by diluting blood with normal saline while suctioning it from the surgical field. Although immediate hemolysis due to suctioning was not very high, the red blood cell damage from suctioning produced by a dynamic blood–air interface might adversely affect the efficiency of cell salvage. (Anesth Analg 2007;104:684 –7) T here are many benefits of autologous blood conservation, including reduction of demands for allogeneic blood (1), avoiding the costs of blood products, avoiding the immunosuppressive effects of allogeneic transfusion (2), reduced incidence of transfusion-related acute lung injury (3), and reduced risk of transfusiontransmitted diseases (4). Several strategies can be applied to avoid allogeneic transfusion. The primary techniques involve preoperative erythropoietin administration and iron supplementation, preoperative autologous donation, acute normovolemic hemodilution, and the application of cell salvage systems. Of these techniques, cell salvage offers the greatest ability to avoid transfusion if applied optimally (5). In an attempt to optimize the capture and return of red blood cells (RBCs), many cell salvage programs minimize the suction pressure used to aspirate blood from the surgical field (6). Minimizing the suction pressure reduces the mechanical injury to RBCs, From the *Department of Anesthesiology, Magee Womens Hospital of University of Pittsburgh Medical center; Departments of †Bioengineering and ‡Pathology, §The Institute for Transfusion Medicine; and 储McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA. Accepted for publication November 21, 2006. Address correspondence and reprint requests to Jonathan H. Waters, MD, Department of Anesthesiology, Magee Womens Hospital of University of Pittsburgh Medical Center, 300 Halket St., Suite 3510, Pittsburgh, PA 15213. Address e-mail to [email protected]. Copyright © 2007 International Anesthesia Research Society DOI: 10.1213/01.ane.0000255208.96685.2e 684 which is mostly due to air bubbles mixing with the blood in the suction cannulae and the tubing connecting the surgical site with the salvage device. The air aspirated with blood during suctioning produces fastmoving bubbles, which expand and collide in the negative pressure environment, generating mechanical stress on the RBCs. Aspiration of air along with blood is a significant cause of RBC damage during processes other than cell salvage. Entrained air during cardiotomy suction damages blood during extracorporeal circulation (7). The blood-air interface contributes to hemolysis in artificial organs (8). Air damages blood in bubble oxygenators as the result of the interfacial denaturation of plasma proteins (9). This causes both sublethal RBC damage (10) and hemolysis of the RBCs (11,12). In this study, an in vitro model was developed to mimic the trauma sustained by RBCs during the suctioning. We measured the effect of suction tip immersion in blood when compared with suctioning from a flat surface to determine ways in which the mechanical injury might be minimized. METHODS On 5 successive days, 11 U of blood group A, citrate phosphate dextrose preserved and anticoagulated whole blood, collected from volunteer donors, was obtained from the blood bank. Informed consent for use of the blood for research purposes was obtained at the time of donation, IRB approval was not required. Vol. 104, No. 3, March 2007 For each experiment, 2–3 U of same type whole blood were mixed in a 2-L beaker with a plastic covered metal stirring rod and kept continually mixed using a magnetic stirrer (Thermolyne, Fisher Scientific, Pittsburgh, PA). For each experiment, the age of the pooled blood was identical. Before commencing the experiments, a sample of the blood was taken for analysis of plasma-free hemoglobin (plfHb) to measure the baseline degree of hemolysis present in the mixed blood. Sixty milliliters of blood was taken from the beaker and placed in a separate small beaker meant to simulate blood drawn from a deep wound. Similarly, 60 mL of blood was placed onto a flat tray covered with a textured plastic material. This was intended to replicate a flat surgical site surface. The blood was then suctioned from either location using a standard Yankauer suction catheter tip (Medi-Vac Yankauer Suction Handle, Catalog K87, Cardinal Health, McGaw Park, IL). The blood was collected into a patient suction canister. The tubing connecting the catheter and a canister was 3 m long with a 6-mm internal diameter (Medi-Vac nonconductive suction tube, Cardinal Health). Suction pressure was regulated using the regulated suction from a COBE BRAT cell salvage device (Arvada, CO). Two suction pressures (100 and 300 mm Hg) were used. The amount of hemolysis induced by suctioning was calculated for each pressure. Suction pressure on the machine was calibrated by the manufacturer during regular machine servicing. Each experiment was performed in duplicate. In addition, 60 mL of blood was mixed with 60 mL of normal saline and suctioned at 300 mm Hg from both simulated surgical sites. After each run, the flow system was thoroughly washed with saline, and the patient suction canister was replaced. From the collected blood, a 5 mL sample was placed into a 7-mL red top vacutainer tube and centrifuged at 3600 rpm for 20 min at room temperature. Blood hematocrit (HCT) was measured (Hematocrit Centrifuge, IEC Clay Adams) in each sample before centrifugation. The supernatant from each sample was transferred to a 1.5 mL microcentrifuge tube and recentrifuged at 14,000 rpm for 15 min at room temperature (Eppendorf 5417R Centrifuge, Hamburg, Germany). The supernatant was then transferred to a spectrophotometer cuvette (1.5 mL semimicro-UV methacrylate cuvette, Fisher Scientific, Pittsburgh, PA) and the plfHb was determined in mg/dL by measuring light absorbance of the samples at 540 nm (Spectronic Genesys 5 Spectrophotometer, Spectronic Instruments, Columbus, OH). The difference between plfHb in the baseline sample and the suctioned sample represented an increase in plfHb (hemolysis) due to exposure to mechanical stress from the suctioning process. As the blood samples had different HCT values, and as mechanical hemolysis depends on HCT, the measured values of plfHb, including those in the baseline samples, were normalized to the standard HCT value of Vol. 104, No. 3, March 2007 40%. This normalization was justified by previous findings (13), which showed that, in the range of HCT values in our samples, there is a linear relationship between levels of mechanical hemolysis and concentration of RBCs. This procedure allowed for comparing blood damage among blood samples with HCT values that were significantly different (i.e., after dilution of blood 1:1 with saline). Normalization was performed with the following equation: Normalized plfHb ⴝ measured plfHb/(measured HCT/40) Finally, the percent of hemolysis was calculated using the formula % Hemolysis ⴝ [plfHb/wblHb] ⴛ 100 where wblHb ⫽ whole blood total Hb concentration in mg/dL. Differences in hemolysis among samples were compared using a one-way analysis of variance with a Bonferroni’s multiple comparison test. A P value of ⬍0.05 was considered statistically significant. Correlation between the age of the blood and the resulting hemolysis was performed with Pearsons correlation test. RESULTS Five sets of experiments were performed. All blood units were type A with ages of 35 days (4 U), 16 days (2 U), 21 days (3 U), and 24 days (2 U). Hemolysis data from the pooled experiments under each set of conditions are presented in Table 1. For all of the tested blood collection conditions the r value correlating the blood age and change in plfHb was 0.84 (P ⫽ 0.16) for the 100 mm Hg suction from a surgical surface, 0.76 (P ⫽ 0.24) for the 300 mm Hg section from a surgical surface, 0.87 (P ⫽ 0.14) for the 100 mm Hg suction from a beaker, and 0.73 (P ⫽ 0.27) for the 300 mm Hg from a beaker. The results are presented after recalculation of plfHb to a standard HCT level (HCT ⫽ 40%) with the assumption that hemolysis is linearly proportional to the concentration of RBCs. As shown in Table 1, hemolysis was always significantly higher when blood was aspirated from the flat surface, regardless of the suction pressure. At a negative suction pressure of 300 mm Hg, dilution of blood with saline (1:1) reduced hemolysis by approximately 55% after suction from the beaker and by more than 60% after collection from the flat surface. Table 2 shows how the suctioning speeds differed based on suction pressure and suction site. As would be expected, 300 mm Hg suction pressure allowed for more rapid suctioning. However, the site of suction was more important: suctioning from a bowl was much more rapid than suctioning from a flat surface. A reduction of blood viscosity from saline dilution reduced the time of © 2007 International Anesthesia Research Society 685 Table 1. Relative Hemolysis as a Function of Surface, Suction Pressure, and Dilution Tests Increase in plasma-free hemoglobin from baseline (mg/dL) Relative hemolysis (%) Surgical surface at 100 mm Hg† (n ⫽ 10) Surgical surface at 300 mm Hg* (n ⫽ 10) Surgical surface at 300 mm Hg ⫹ dilution (n ⫽ 10) Beaker at 100 mm Hg (n ⫽ 10) Beaker at 300 mm Hg (n ⫽ 10) Beaker at 300 mm Hg ⫹ dilution (n ⫽ 10) 135.5 ⫾ 35.9 304.3 ⫾ 60.6 119.3 ⫾ 15.0 36.0 ⫾ 12.8 63.9 ⫾ 15.2 27.7 ⫾ 8.6 1.02 ⫾ 0.27 2.29 ⫾ 0.46 0.90 ⫾ 0.11 0.27 ⫾ 0.10 0.48 ⫾ 0.11 0.21 ⫾ 0.06 Data presented as Mean ⫾ SEM. * Surgical surface at 300 mm Hg significantly different than all other groups (P ⬍ 0.001). † Surgical surface at 100 mm Hg is significantly different from beaker at 100 mm Hg (P ⬍ 0.05) Table 2. Suction Speed Time of suctioning (s) Testsa Surgical surface at 100 mm Hg* Surgical surface at 300 mm Hg† Surgical surface at 300 mm Hg ⫹ dilution‡ Beaker at 100 mm Hg Beaker at 300 mm Hg Beaker at 300 mm Hg ⫹ dilution 75.3 ⫾ 16.4 (60 ml) 42.1 ⫾ 12.7 (60 ml) 32.0 ⫾ 5.7 (120 ml) 12.6 ⫾ 6.0 (60 ml) 4.8 ⫾ 0.8 (60 ml) 4.3 ⫾ 0.8 (120 ml) a n ⫽ 10 for each condition. * Surgical surface at 100 mm Hg significantly different than all group comparisons (P ⬍ 0.001). † Surgical surface at 300 mm Hg significantly different that beaker at 100 mm Hg, beaker at 300 mm Hg, and beaker at 300 mm Hg ⫹ dilution (P ⬍ 0.001). ‡ Surgical surface at 300 mm Hg ⫹ dilution significantly different than beaker at 100 mm Hg, beaker at 300 mm Hg, and beaker at 300 mm Hg ⫹ dilution (P ⬍ 0.001). suction from both a beaker and the flat surface by approximately 60%. DISCUSSION Hemolysis ranged from 0.21% to 2.29% in this study. In the only other study of this topic, Gregoretti (6) reported hemolysis ranging from 0.32% ⫾ 0.21% when blood alone was aspirated to 2.85% ⫾ 0.22% (P ⬍ 0.05) when a blood–air admixture was suctioned at a vacuum pressure of 300 mm Hg, similar to the values in our study. Mathematical modeling of cell salvage has revealed that small changes in RBC processing efficiency can make large differences in the maximum allowable blood loss that a patient can sustain before allogeneic transfusion therapy (14,15). These models suggest that a 70 kg patient with a starting HCT of 45% can sustain a blood loss of 9600 mL if a transfusion trigger of 21% is used and cell salvage captures 60% of lost RBCs. The sustainable blood loss increases to 13,750 mL if 70% RBC recovery is achieved. Thus, small changes in RBC recovery can result in large differences in the ability to avoid allogeneic transfusion. In an attempt to maximize capture of RBCs, many cell salvage programs will minimize suction pressure applied to the RBCs when it is being removed from the surgical field. Decreasing the suction pressure decreases the shear forces applied to the RBCs, which in turn decreases hemolysis. In addition, surgical 686 Minimizing Mechanical Blood Damage sponges are rinsed with saline before discard with the rinse solution being processed through the cell salvage cycle, resulting in capture of these RBCs. Regulation of suction pressure and rinsing of sponges are the only known techniques for maximizing efficiency of RBC recovery. The visual appearance of the blood after high suction from the flat surface showed heavy amounts of frothing from aspirated air. Diluting blood with normal saline reduced the frothing and mechanical stress, resulting in an approximately 60% reduction in hemolysis. This suggests that hemolysis can be reduced in some surgical environments by instilling normal saline into the surgical field during suctioning. For example, spine surgery involves a small suction tip on a relatively flat, shallow surgical field. In these procedures, use of saline along with suctioning would facilitate RBC recovery. The same effect might be achievable by combining normovolemic hemodilution with cell salvage. The highest physiological shear stress in the normal vasculature is about 10 N/m2 (16). The highest wall shear stresses applied to blood in our experiments were about 20 N/m2. This shear stress is significantly lower than those of 200 –300 N/m2 considered to be lethal for RBCs (17). This suggests that other factors are more important than the aspiration method in the RBC loss during blood salvage. The blood–air interface may also contribute to increased hemolysis during filtration at the point of the collection, and the concentrating and washing processes. Sublethal RBC damage is analogous to “accelerated” RBC aging, and promotes early removal of the transfused RBCs from the vascular system (18). Our results confirm the previous work of Gregoretti, which showed that suction of air should be avoided to enhance RBC capture and return. The degree of hemolysis observed in both studies does not explain the large differences in capture rates obtained in Waters’ mathematical modeling (16). The reason for this could be a sublethal blood damage produced during the first stage of blood moving from a surgical site to its final mixing with saline before it is returned to a patient. Sublethal damage, which is not easy to identify, could significantly reduce RBC survival during washing and filtration. Minimizing suction pressure will decrease ANESTHESIA & ANALGESIA sublethal damage and increase the efficiency RBC salvage. REFERENCES 1. Wallace EL, Churchill WH, Surgenor DM, et al. 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