Cardiopulmonary bypass (CPB) is commonly used to support the circulation during operative repair of acquired and congenital heart diseases. However, CPB is associated with certain pathophysiologic side effects that are related to the time on bypass and the complexity of the underlying operation. Systemic inflammation, hemolysis, and coagulation dysfunction have all been described as major complications.1–3 Many pathways and factors contribute to these CPB-associated morbidities and include contact of the blood components with artificial surfaces, air exposure, ischemic–reperfusion injuries, operative trauma, and suction.4 Much research has centered on the causes of systemic inflammation, coagulation abnormalities, and hemolysis during CPB and cardiotomy suction (CS) use, but the exact cause of these side effects is not completely understood.
Cardiotomy suction allows better visualization of the operative field by returning shed blood back to the heart–lung machine using negative pressure applied to a suction wand or device. However, CS creates dynamic conditions that expose blood to air, suction, and additional exposures to plastic surfaces. These factors may cause a pronounced inflammatory response, hemolysis, and activation of certain coagulation and humoral factors in blood that has been exposed to conditions created by CS.5 Reinfusion of CS blood may contribute to secondary organ dysfunction and other complications including renal failure, respiratory failure, neurologic dysfunction, bleeding disorder, and myocardial injury.6,7 Eliminating CS may reduce these side effects; however, CS is required for many cardiovascular procedures, including complex intracardiac and great vessel procedures as well as valve replacements and congenital repairs. Avoiding the use of CS may increase the need for homologous blood transfusions postoperatively.5,8
Previously, this laboratory described an in vitro red cell damage model where room air, suction, and a combination of the air and suction were exposed to static blood samples.9 In both sheep and human blood, hemolysis was dose related to the amount of suction and air flow in combination when tested together. The maximum test conditions were −600 mm Hg (suction) and 100 ml/min (air flow). Isolated exposure to either air or suction alone showed minimal changes in hemolysis. The current study was designed to study to effects of these conditions on platelets and leukocytes.
Materials and Methods
After the approval from the Institutional Internal Review Board and subject consent, 20 ml of fresh blood from 23 healthy human volunteers (n = 23 for platelets, n = 10 for leukocytes) was individually acquired through venipuncture and transferred into citrated tubes (BD Lavender Vacutainer Sodium Citrate 0.109M, 3.2%; Becton Dickinson, Franklin Lakes, NJ) and used to conduct the experiment at room temperature (24–26°C). Five milliliter aliquots from each sample was individually transferred into 50 ml polypropylene centrifuge tubes (cat no. 06-443-21; Thermo Fisher Scientific Inc., New York, NY) that were tested in four different and unique conditions.
In Vitro Cardiotomy Suction Model Apparatus
The experiment was tested on an apparatus consisting of a test chamber made from two modified and connected 50ml polyethylene centrifuge tubes (Thermo Fisher Scientific Inc.), a vacuum pump used to generate a suction −600 mm Hg (Model ME4; Vacuubrand Inc., Essex, CT), and a Matheson 602 series precision gas flow meter (Matheson Tri-Gas, Montgomeryville, PA) to control a consistent 100 ml/min room air flow through blood sample. These conditions were selected because they elicited maximal red cell damage in previous work and allowed for comparison with similar test conditions. The gas flow meter was connected to an air line with the tip positioned just above the bottom of the test chamber. As air bubbles traveled through the blood sample, an air–blood interface was generated and a blood–air bubble column expanded from the base of the test chamber. The air–blood column was contained within the test chamber by a polyurethane sponge plug coated with Antifoam A (Sigma-Aldrich, St. Louis, MO) and a rubber stopper. The Antifoam A sponge isolated the reaction to the test chamber by debubbling the foam that was created by the reaction to air. The pressure within the circuit was monitored continuously with a digital pressure indicator (Model DPI 705; Omega Engineering Inc., Stamford, CT). All experiments were conducted at room temperature (Figure 1).
Blood from each sample was tested in four different conditions (Table 1); each condition was applied individually for 10 minutes in time. Group A consisted of baseline conditions (control). Suction at −600 mm Hg was applied to the blood sample and designated as group B, which was generated and regulated by the vacuum pump. Group C blood was exposed to air at 100 ml/min, generating a frothing blood–bubble mix. Group D was a combination of air and suction exposure described in groups B and C. The specific air and suction settings were chosen because they elicited maximal hemolysis in previous work and they represent conditions that can occur during the clinical use of CS.9 One physical factor that does exist in all blood and flow models are shear forces. Models do exist for calculating shear; however, there was no practical way to calculate shear in this test model.
Supplemental Antifoam A Testing
The potential did exist for an artifact related to the exposure of blood to Antifoam A to activate the blood independent of the individual test conditions. Additional pieces of Antifoam A–coated sponge used in this test model were taken from a commercially available cardiac bypass reservoir (Terumo, Tokyo, Japan) and added to samples of blood from each group to determine whether polyurethane Antifoam A caused activation. Surface marker expression was then quantified.
After running the different experimental conditions, 3 ml of blood from each aliquot was analyzed to quantify hemolysis. The measurement of plasma-free hemoglobin (PFHb) was made using a spectrometer with absorbent readings at 577, 560, and 593 nm.10
Indirect Immunofluorescence Flow Cytometry Preparation
Monocyte and granulocyte CD11b expression was used as a marker for leukocyte activation. In addition, CD62P (P-selectin) expression was measured on platelets as a marker of platelet activation.
The conditioned blood aliquots were diluted from 10 to 990 μl with Hank’s Balanced Salt Solution without CaCl2, MgCl2, MgSO4, and phenol red in polypropylene 12 × 75 tubes and gently mixed. In addition, 100 μl of the diluted blood was added to another two labeled tubes (P1 and P2). As a control, 10 μl of fluorescein isothiocyanate (FITC)-mouse IgG1 and 10 μl of PE-mouse IgG1 were added to P1 tube and for the experimental tube, 10 μl of CD61-FITC and 10 μl of CD62P-PE were added to P2 tube. Then, both tubes were incubated at room temperature in dark for 15 minutes. Later on, 700 μl of 1% formaldehyde buffer (1% formalin in Dulbecco’s phosphate buffered saline [dPBS]; Gibco, Grand Island, NY) was added to each tube, vortexed, and transferred to 12 × 75 polystyrene tubes and stored for 24 hours in refrigerator at 4°C. Samples were then run using a flow cytometer, and data were collected and analyzed.
For leukocyte CD11b measurement, 100 μl of the conditioned aliquot was transferred individually to polystyrene tubes (W1) and directly labeled with 10 μl of antibody for PE-mouse antihuman CD11b and FITC-mouse anti-pig CD14. As controls for FACS analysis, 10 μl of FITC-mouse IgG2b negative control and PE-mouse IgG2a negative control were used in the other tube (W2). Once stock solutions were applied, tubes were stirred and incubated in the dark at 4°C for 30 minutes. Two milliliters of BD RBC lysis buffer was added to each tube and incubated for 10 minutes at room temperature in the dark. Tubes were then centrifuged for 5 minutes at 1,100 rpm. After the removable of supernatant, 2 ml of washing solution buffer (dPBS + 0.1% sodium azide+ 0.5% BSA) was added to each tube. Tubes were centrifuged a second time for 5 minutes at 1,100 rpm. After the removal of supernatant, 250 μl of 1% formaldehyde buffer was added to each tube. Tubes were vortex mixed, transferred to 12 × 75 polystyrene tubes, and stored for 24 hours in a refrigerator at 4°C.
For cell counts, the surface expression of CD14 on the leukocyte was used to approximate the white blood cell count. The surface expression of CD61 on platelets was used as a measure of platelet count.
To account for variances between human subjects, all measured parameters were compared as a percentage of baseline with values obtained after each test condition. Baseline measurements for each marker displayed a nonparametric distribution; therefore, the nonparametric Wilcoxon signed-rank test was used to determine statistical significance for surface marker expression. Data were compared with baseline values for each blood sample and presented in percentile value where baseline was 100%. Because multiple variables were being tested, the Bonferroni correction was applied. A significant p value was defined as p < 0.0125.
The current study compares the effects of air exposure and suction on blood cell activation and hemolysis using an in vitro CS model. In this model, the air–blood interface caused leukocyte activation and hemolysis.
Whole blood hemolysis was significantly higher in groups C and D, corroborating previous studies.9 The mean PFHb in groups A and B were similar (p = 0.741). In groups C and D, PFHb increased to an average of 50.55 ± 2.91 mg/dl (p < 0.001) and 99.59 ± 4.53 mg/dl (p < 0.001), respectively (Figure 2).
Leukocyte and Platelet Counts
The leukocyte and platelet counts showed no difference between control and any experimental condition. There were no statistically significant differences in leukocyte and platelet counts between groups as determined by the Wilcoxon signed-rank test The mean total leukocyte counts compared with baseline were 94 ± 13% (p = 0.655) in group B, 95 ± 14% (p = 0.715) in group C, and 95 ± 9% (p = 0.735) in group D. Platelet counts were similarly unchanged in all conditions with average of 105 ± 34% (p = 0.5130) in group B, 105 ± 24% (p = 0.466) in group C, and 116 ± 36% (p = 0.860) in group D (Figure 3).
Inflammatory Response Markers
The results showed an increase in the expression of CD11b in human granulocytes with a mean of 304 ± 230%, (p = 0.0029) in group C and a mean of 232 ± 169% (p = 0.0123) in group D. No significant changes occurred in group B with mean of 116 ± 35% (p = 0.7623) (Figure 4).
The expression of CD11b in monocytes showed no significant changes, group B with mean 110 ± 26% (p = 0.9214), group C with mean 126 ± 28% (p = 0.51), and group D with mean 114 ± 25% (p = 0.6926) (Figure 4).
The expression of P-selectin was not significantly different from baseline in groups B (99 ± 30%, p = 0.9633), C (147 ± 68%, p = 0.0166, and D (122 ± 44%, p = 0.2103) (Figure 4).
Supplemental Antifoam A Testing Result
Samples of group A blood with no additive showed no significant difference in granulocyte, monocyte, or platelet activation compared with group A blood exposed to Antifoam A covered polyurethane; p = 0.439, p = 0.1130, and p = 0.0303, respectively.
This study evaluated the effect of air and suction on leukocytes and platelets in a simple in vitro model. The air–blood interface used in this test model with and without suction caused leukocyte activation and hemolysis, which confirmed earlier studies from this laboratory that the combination of air and negative pressure causes hemolysis.9 The goal was to evaluate the different effects of air exposure and suction independently, and in combination on blood components and how this may contribute to leukocyte activation and hemolysis. Air exposure caused activation of granulocytes, but not monocytes or platelets.
In this study, blood samples were anticoagulated with citrate, which may have had a modulating effect on leukocyte and platelet activation. Further studies will be conducted with heparin anticoagulation to determine any effects that occur as a result of using different anticoagulants. It is possible that heparin alone can activate an inflammatory pathway or the chelation of calcium ion by citrate may decrease activation.11
Cardiotomy suction has been recognized as a major source of hemolysis during cardiac operations.12 Cardiotomy suction may play a significant role in inducing a systemic inflammatory response.5 The influence of CS on postoperative systemic inflammatory response is not fully understood. Many studies have demonstrated elevated levels of hemolysis and inflammatory mediators in CS blood and therefore concluded that retransfusion of CS blood contributes to the postoperative response.13 However, these studies do not clarify the cause of this activation.
Measuring granulocyte and monocyte CD11b expression provide a means to identify an early-onset inflammatory response.14 Data exist that show an increase in pro-adhesion potential, as indicated by an increase in granulocyte and monocyte CD11b.15 In this study, the results indicate that granulocyte CD11b expression was increased during isolated air–blood interface and combined air-suction conditions. On the contrary, no changes in the expression of CD11b were observed in isolated suction condition alone. These data support the idea that the inflammatory response caused by CS may be related to the excess amount of air exposure but not isolated suction. Granulocyte activation causes a release of cytokines, which mediate the inflammatory response. In future studies, this model will be used to measure the cytokine levels and correlate these with CD11b expression.
Cardiopulmonary bypass induces a degree of hemolysis as a result of a combination of mechanical trauma and biological mechanisms. This study shows that the source of hemolysis is primarily from factors associated with an air–blood interface and suction, as have been previously described.9 The data describe a significant increase in PFHb in isolated air–blood interface, even when combined with suction. This observation corroborates other investigations that reported an increase in red blood cell damage during the use of CS.12,16
Cardiotomy suction could cause platelet damage or activation due to aspiration of air and blood. Excessive use of CS may result in a decreased ability of platelets to aggregate.17 P-selectin (CD62P) is a component of the α-granule membrane of resting platelets that is expressed only on the platelet surface during and after platelet degranulation and secretion.18 Platelet surface P-selectin is considered to be the “gold standard” marker of platelet activation.19 Despite the fact that many studies have presented alterations in platelet count, clot formation, and platelet activation during CPB,20 the results from this study did not indicate any statistically significant increase in P-selectin expression or any significant change in platelet count under any test condition. Platelet function was not evaluated, but function will be measured in future experiments.
There are several limitations to this study. An in vitro test does not clearly reproduce the in vivo setting in which variations in patients or additional factors such as temperature, shear forces, and other artificial organs such as filters and the gas exchange device may induce either minor or profound changes. A negative pressure of −600 mm Hg and 100 ml/min of air flow used may be considered excessive for the length of time (10 minutes) in which the blood was tested. These conditions are routinely reached or exceed during clinical CPB. Further studies will vary the amount of suction and air to determine whether a “dose-response” effect on leukocytes exists. The experimental model evaluated static blood; thus, the application of these parameters in an active blood flowing model would be a better representation of in vivo use.
In clinical application, an air–blood interface is not only observed in CS but also observed when intermittent or continuous streams of air siphon down the venous line toward the venous reservoir. The amount of air is related to the siphon pressure or the amount of air being entrained at the venous cannulation site in the heart. Although this air is removed in the venous reservoir, it is certainly possible that the air–blood interface could enhance cellular activation or injury. Minimizing air entrainment in the venous line or eliminating the air–blood surface in the venous reservoir could minimize blood damage.
The findings from this study indicate that air exposure and suction during simulated CS is a cause of hemolysis and granulocyte activation. The response could be minimized by reducing air exposure to the blood and minimizing suction pressures. The clinical use of CS varies widely among individual practitioners; yet, it is widely accepted that blood damage occurs, especially when there are large amounts of suction air, foam, and blood. “Sucker bypass” is one such extreme when massive amounts of air, blood, and suction are used in an emergent setting of massive hemorrhage. It is certainly feasible that shed blood from the operative field can be discarded or salvaged by a red cell washing device. However, immediate retransfusion is necessary if there is large blood loss from the operative field. When a coronary suction tip becomes occluded, an extreme negative pressure can be created, which can meet or exceed the values tested in this model. A pressure limited suction wand that vents in atmosphere would reduce extremes in negative pressure if the wand became occluded. In addition, large amounts of air are exposed to blood if the suction pump is on and blood is contained in the suction tubing. Hemolysis and granulocyte activation caused by CS could be minimized by limiting air exposure and suction pressure.
The authors acknowledge the efforts of Jacob Miller and Salvatore Aiello for their technical assistance, Dr. Gail Annich for correspondence with the Institutional Review Board at the University of Michigan, Dr. Alvaro Rojas-Pena and Cindy Cooke for review and preparation of the manuscript, and the Undergraduate Research Opportunity Program (UROP) at the University of Michigan.
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leukocytes; hemolysis; inflammatory mediators; cardiopulmonary bypass; platelets