Biocompatible evaluations by various methods have been reported 1–5; however, there are no papers describing discriminating evaluation of biocompatibility of oxygenators. Some researchers have focused on complement activation products as well as platelet counts during in vitro studies 2,3 or in clinical cases, 4,5 and activation of the complement cascade has been suggested as an index of biocompatibility. However, hemolysis is one of the most critical blood reactions of blood contact with circulatory assist devices. 6,7 In clinical cases, especially in the case of long-term extracorporeal membrane oxygenation (ECMO), biocompatible performance can be estimated by using degree of hemolysis. 8
A specialized hemolysis test protocol for oxygenators was developed, 9 and the hemolytic characteristics associated with oxygenators during experimental conditions that simulate their use in cardiopulmonary bypass (CPB) were previously reported by our group. 10 At this time, a comparative study was performed after this protocol to determine the hemolytic characteristics of the clinically available oxygenators during ECMO and the relationship between these hemolytic characteristics and the pressure drop in the blood chamber was examined.
Materials and Methods
The hemolytic characteristics of three types of oxygenators, Menox EL4000 (Dainippon Ink and Chemicals Inc., Tokyo, Japan), Module 4000 (Dideco, Mirandola, Italy), and Mera Exelung Binding HPO-15H (Senko Medical Instrument Co. Ltd., Tokyo, Japan), were evaluated using fresh bovine blood. These are all clinically available polyolefin or polypropylene oxygenators for ECMO or pediatric CPB usage. The priming volumes are 110 ml, 140 ml, and 260 ml, respectively, with membrane surface areas of 0.8 m2, 1.0 m2, and 1.5 m2. It has been reported that the gas and biocompatible performance of these oxygenators is excellent 11; however, these three oxygenators are not available in the United States.
Blood was drawn from healthy donor calves per phlebotomy into a blood bag containing 63 ml of citrate phosphate dextrose adenine solution. The blood came from a single calf species (Dexter strain) from the same ranch. Before pumping, the blood was examined to eliminate damaged samples (having clots detected by visual inspection, partial hemolysis detected by measuring plasma free hemoglobin, high or low hematocrit levels, etc.). The blood was refrigerated during shipment and upon arrival in our laboratory. All of the units of blood were used within 72 hours of acquisition.
According to the established method in this laboratory, 9,10 a mock circuit was assembled to measure hemolysis (Figure 1). Each circuit contained a pump, oxygenator, appropriate polyvinyl chloride tubing, and connectors with stopcocks for blood sampling and pressure measurements. The studies adhered to the recommended practice for assessment of hemolysis as described by the American Society of Testing and Materials. 12 The oxygenator was placed on the outflow side of the centrifugal pump within 6.6 feet (2 m), with a 3/8 inch (9.5 mm) inner diameter polyvinyl chloride tubing loop. A polyvinyl chloride reservoir with a sampling port was attached, and the Gyro C1E3 (Kyocera Corp., Kyoto, Japan) was used for the centrifugal pump. The circuit was rinsed with saline and then flushed with CO2 to eliminate microbubbles before it was filled with the prewarmed blood. The blood flow was fixed at 1 L/min as in an ECMO condition. The gas flow was also fixed at 1 L/min. The blood flow was monitored with an ultrasonic flowmeter (Transonic System T108, Ithaca, NY). A screw clamp established a pressure head of 100 mm Hg. To maintain the temperature at 37 ± 2°C, two water reservoirs were connected to the heat exchanger, and the blood reservoir was put between these water reservoirs. Control samples were taken 5 minutes after the mock circulation loop was activated, after which the samples were taken every 20 minutes up to 120 minutes. The plasma samples were centrifuged (5 minutes; 1,500 rpm), and free hemoglobin assay was conducted. Plasma free hemoglobin concentration was measured using a 3,3′,5,5′-tetramethylbenzidine (TMB) colorimetric assay (Sigma Diagnostic Kit No.527, St. Louis, MO).
Calculation of Normalized Index of Hemolysis
Blood damage was expressed as a Normalized Index of Hemolysis (NIH), according to the equation of Koller and Hawrylenko:13MATHwhere ΔfHb is the increase in free plasma hemoglobin concentration (g/L) during the testing period, Δt is the testing time (minutes), Ht is the hematocrit (%), V is the blood volume of each circuit (L), and Q is the flow rate expressed as L/min. ΔfHb/Δt was obtained from the slope of the regression line between the free plasma hemoglobin and the test time. The sampling times were plotted on the X-axis, and the free plasma hemoglobin values were plotted on the Y-axis. The slope of the regression line between them was derived from the graph using computer software. The obtained slope indicated ΔfHb/Δt.
Definition of Normalized Index of Hemolysis for Oxygenators
The Normalized Index of Hemolysis for Oxygenators (NIHO) has been modified according to the American Society of Testing and Materials standards. 12 The NIH value, which was obtained from the circuit without an oxygenator, was subtracted from the primary NIH value, which was obtained from the circuit with an oxygenator, to eliminate the effects of the centrifugal pump or other artifacts. This was defined as the value of NIHO. 9,10
Pressure Drop Measurement
The blood pressure drop in the circuit was also monitored. Absolute pressure values from the blood chamber were measured while testing the oxygenator at blood flow rates raised incrementally from 0.5 L/min to the maximum flow for each oxygenator. To accurately measure the pressure drop, independent pressure measuring ports were installed in the blood inlet and outlet sides of the oxygenator.
All values were expressed as mean ± standard deviation. Comparisons between the three oxygenators were made using a Kruskal-Wallis test. Hemolysis and pressure drop studies were repeated three times, and the averages were calculated and plotted. Values of p < 0.05 were considered to be statistically significant.
NIHO of each oxygenator at a blood flow rate of 1 L/min is shown in Table 1. The Menox demonstrated the best hemolytic performance characteristics, with the lowest NIHO value (0.0070 ± 0.0009), and increased from Menox to Dideco (0.0113 ± 0.0099) to Mera (0.0164 ± 0.0043). However, there were no significant differences among the oxygenators.
The pressure drop measurements are shown in Figure 2. The lowest pressure drop on the blood passage side across the membrane oxygenator was also shown by Menox and increased from Menox to Dideco to Mera. Mean pressure drop at a blood flow rate of 1 L/min was 3 mm Hg in Menox, 32 mm Hg in Dideco, and 44 mm Hg in Mera. Similar to the results under CPB conditions (5 L/min), the NIHO value has a close relationship to the pressure drop.
Today in the United States, ECMO use is increasing annually because of the excellent clinical results. 14,15 Although ECMO was established as a treatment for respiratory failure, it has problems, such as blood trauma, due to blood contact in all circulatory assist devices, and hemolysis remains one of the most serious problems during ECMO. 8 This is usually not a problem during routine CPB, but it may be an issue during ECMO for respiratory assistance, when the patient may depend on the oxygenator for continuous gas exchange for days or weeks. 16 However, the hemolytic characteristics associated with oxygenators for ECMO are not well defined. Different types of oxygenators cause different amounts of hemolysis based on shear stress and blood exposure time. In this study, following a specialized hemolysis test protocol for oxygenators established by our group, three types of clinically available oxygenators were evaluated under ECMO condition.
Dideco Module 4000 and Mera HPO-15H are the conventional microporous polypropylene oxygenators, and the Menox EL4000 is a polyolefin hollow fiber membrane oxygenator with a double layer structure (asymmetric structure). This membrane has blind ended micropores on the membrane surface and can effectively prevent plasma leakage through the pores. The oxygenator using this membrane has been reported to be useful in long-term extracorporeal respiratory support. 17 In this study, although there were no significant differences among the oxygenators, the Menox EL4000 demonstrated the best biocompatible performance characteristics. Similar to our previous report, the NIHO value also has a close relationship to the pressure drop under ECMO conditions. The effect of the pressure drop in an oxygenator and its effect on blood trauma is still a subject of debate. 18,19 Bearss 18 reported a weak correlation between the pressure drop and hemolysis index; however, De Somer et al.19 mentioned no statistical differences.
Senko Medical Instrument has developed a new silicone coated hollow fiber membrane oxygenator. The blood contact surface of this hollow fiber is coated with a 0.2 μm ultra thin silicone layer. It has been determined to have good biocompatibility and gas transfer performance characteristics. 2,4,20,21 In addition, a heparin coated hollow fiber oxygenator was introduced, and its usefulness was reported. 3,5,22,23 Hemolysis occurs after mechanical damage to erythrocytes and by complement mediated erythrolysis. 24 The primary physical forces applied to blood cells in an oxygenator are shear stress, pressure, wall impact, and surface phenomena. Although we did not evaluate the silicone or heparin coated oxygenators at this time, it will be interesting to evaluate their hemolytic characteristics according to our protocol.
There were limitations of the in vitro system used in this study, which had a short experimental time and a low pressure head from a screw clamp. This circuit includes an oxygenator that affects the fragility of red blood cells. Therefore, hemolysis was observed earlier and we took blood samples every 20 minutes up to 2 hours. However, the measured data had a considerable scatter and large standard deviations, whereas the real values were so small that it was difficult to see statistically significant differences among the oxygenators. Compared with CPB (5 L/min), 10 measurements under ECMO conditions (1 L/min) may be affected easily by other artifacts due to low blood flow. Further improvements (including longer experimental periods, higher pressure heads, etc.) are necessary to accurately evaluate the devices under a low blood flow condition.
Similar to the previous report during CPB conditions, the hemolytic characteristics of the oxygenators have a close relationship to the pressure drop during ECMO. This new evaluation method established by our group is also applicable to comparison of the biocompatibility performance of different types of clinically available oxygenators for ECMO usage.
1. Niimi Y, Yamane S, Yamaji K, Tayama E, Sueoka A, Nosé; Y: Protein adsorption and platelet adhesion on the surface of an oxygenator membrane. ASAIO J 43: M706–M710, 1997.
2. Watanabe H, Hayashi J, Ohzeki H, Moro H, Sugawara M, Eguchi S: Biocompatibility of a silicone-coated polypropylene hollow fiber oxygenator in an in vitro
model. Ann Thorac Surg 67: 1315–1319, 1999.
3. Videm V, Mollnes TE, Garred P, Svennevig JL: Biocompatibility of extracorporeal circulation: In vitro
comparison of heparin-coated and uncoated oxygenator circuit. J Thorac Cardiovasc Surg 101: 654–660, 1991.
4. Shimono T, Shomura Y, Tani K, et al: Clinical evaluation of a silicone coated hollow fiber oxygenator. ASAIO J 43: M735–M739, 1997.
5. Videm V, Svennevig JL, Fosse E, Semb G, Østerud A, Mollnes TE: Reduced complement activation with heparin-coated oxygenator and tubing in coronary bypass operation. J Thorac Cardiovasc Surg 103: 806–813, 1992.
6. Takami Y, Nakazawa T, Makinouchi K, Glueck J, Benkowski R, Nosé; Y: Effect of surface roughness on hemolysis in a centrifugal blood pump. ASAIO J 42: M858–M862, 1996.
7. Kawahito K, Nosé; Y: Hemolysis in different centrifugal pumps. Artif Organs 21: 323–326, 1997.
8. Steinhorn RH, Isham-Schopf B, Smith C, Green TP. Hemolysis during long-term extracorporeal membrane oxygenation. J Pediatr 115: 625–630, 1989.
9. Maeda T, Iwasaki A, Kawahito S, et al: Preclinical evaluation of a hollow fiber silicone membrane oxygenator for extracorporeal membrane oxygenator application. ASAIO J 46: 426–430, 2000.
10. Kawahito S, Maeda T, Yoshikawa M, et al: Blood trauma induced by clinically accepted oxygenators. ASAIO J 47: 492–495, 2001.
11. Nosé Y, Motomura T, Kawahito S (ed): Oxygenator: Artificial Lung: Past, Present, and Future. Houston: ICAOT/ICMT, 2001.
12. Annual Books of Standards (American Society of Testing and Materials). Standard practice for assessment of hemolysis in continuous flow blood pumps. F1841: 1295–1299, 2000.
13. Koller T Jr, Hawrylenko A: Contribution to the in vitro
testing of pumps for extracorporeal circulation. J Thorac Cardiovasc Surg 54: 22–29, 1967.
14. Ichiba S, Bartlett RH: Current status of extracorporeal membrane oxygenation for severe respiratory failure. Artif Organs 20: 120–123, 1996.
15. Von Segesser LK: Cardiopulmonary support and extracorporeal membrane oxygenation for cardiac assist. Ann Thorac Surg 68: 672–677, 1999.
16. Liebold A, Reng CM, Philipp A, Pfeifer M, Birnbaum DE: Pumpless extracorporeal lung assist: Experience with the first 20 cases. Eur J Cardiothorac Surg 17: 608–613, 2000.
17. Tatsumi E, Taenaka Y, Nakatani T, et al: A VAD and novel high performance compact oxygenator for long-term ECMO with local anticoagulation. ASAIO Trans 36: M480–M483, 1990.
18. Bearss MG: The relationship between membrane oxygenator blood path pressure drop and hemolysis: An in-vitro evaluation. J Extra Corpor Technol 25: 87–92, 1993.
19. De Somer D, Foubert L, Vanackere M, Dujardin D, Delanghe J, Van Nooten G: Impact of oxygenator design on hemolysis, sheer stress, and white blood call and platelet counts. J Cardiothorac Vasc Anesth 10: 884–889, 1996.
20. Shimono T, Shomura Y, Tahara K, et al: Experimental evaluation of a newly developed ultrathin silicone layer coated hollow fiber oxygenator. ASAIO J 42: M451–M454, 1996.
21. Niimi Y, Ueyama K, Yamaji K, et al: Effects of ultrathin silicone coating of porous membrane on gas transfer and hemolytic performance. Artif Organs 21: 1082–1086, 1997.
22. Crotti S, Tubiolo D, Pelosi P, Chiumello D, Mascheroni D, Gattinoni L. Long-term evaluation of gas exchange and hydrodynamic performance of a heparinized artificial lung: comparison of two different hollow fiber pore sizes. Int J Artif Organs 20: 22–28, 1997.
23. Muehrcke DD, McCarthy PM, Stewart RW, et al: Complications of extracorporeal life support systems using heparin-bound surfaces. The risk of intracardiac clot formation. J Thorac Cardiovasc Surg 110: 843–851, 1995.
24. Van Oeveren W, Kazatchkine MD, Descamps-Latscha B, et al: Deleterious effects of cardiopulmonary bypass. J Thorac Cardiovasc Surg 89: 888–899, 1985.