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HEMOLYSIS OF BLOOD DUE TO EXPOSURE TO DIFFERENT TYPES OF PLASTIC TUBING AND THE INFLUENCE OF ETHYLENE-OXIDE STERILIZATION
HEMOLYSIS OF BLOOD D U E T O EXPOSURE TO DIFFERENT TYPES OF PLASTIC T U B I N G THE INFLUENCE OF ETHYLENE-OXIDE Teruo Hirose, M.D.,* Robert Goldstein, M.D.,** Charles P. Bailey, M.D.,*** New York, N. Y.
AND
STERILIZATION and
T
HE continuous improvements in the various kinds of equipment available for extracorporeal circulatory bypass and the development of more intricate operations for intracardiac pathologic conditions are reorienting our thoughts in the direction of longer and longer periods of perfusion. Partial circulatory assistance and hemodialysis imply even longer periods of bypass. The anticipated increase in time of exposure of the participate and fluid content of the blood to the various metallic or plastic components of the apparatus inevitably would seem to tend to exaggerate any hemolytic or other deleterious effects. It is generally admitted that such changes in the blood can precipitate serious visceral complications (such as renal or hepatic failure) even though the exact mechanisms of their effects may not be understood fully. Therefore, any contribution to the known methods of limiting or reducing such blood damage should be worth while. Since using the ethylene-oxide gas sterilization technique at the Flower and Fifth Avenue Hospitals in New York City for the preparation of our perfusion equipment, we have observed a significant increase in the incidence of renal complications. This has been associated with consistently higher reported levels of free hemoglobin in the perfused blood (using identical extracorporeal equipment for comparable periods of time). The question was raised whether ethylene-oxide gas might not be absorbed by or combined with the plastic materials used in the circuit, thus changing their nature and their effect on the blood. It was deemed improbable that this gas would combine with or be absorbed by the metallic (steel) or glass components of the equipment. The recent report of Keith and his associates1 concerning the enhanced hemolytic effect upon perfused blood of one batch of plastic tubing of a type commonly used for extracorporeal bypass suggested to From the New York Medical College, Flower and Fifth Avenue Hospitals, 1249 Fifth Ave., New York 29, N. Y. Aided by Grant H 6966 from the 17. S. Public Health Service and American Heart Association. Received for publication April 4, 1962. •Teaching- Fellow in Surgery. **Associate Professor of Medicine. "•Professor of Surgery. 245
HIROSE, GOLDSTEIN, B A I L E Y
246
J. Thoracic and Cardiovas. Suiy.
mg 1, 000
2 hours A
B
A
24 hours B
Pig. 1.—Plasma hemoglobin in different sizes of various plastic tubes (48 hr. after gas sterilization) incubated at 37° C. for 2 hr. and 24 hr. (canine blood).
us that different types and brands of the available plastic materials might have variable effects upon the blood and might be differentially affected by agents which could be absorbed by or might combine with them. Hence, it was decided to carry out a study of the effect upon sterile canine and human blood of exposure for periods up to 24 hours to various types (brands) and sizes of commonly used plastic tubing which had been sterilized, respectively, by steam or ethylene-oxide gas. It was realized at the outset that the contemplated studies would not be directly and fully referable to clinical problems of perfusion since the clinical and experimental conditions are not exactly comparable. In the present study no movement of the exposed blood has been incorporated in the test. It is intended that a later study will be oriented in the direction of evaluating damage due to movement of the blood through the several types of bypass equipment. Furthermore it is obvious that one cannot equate the level of plasma hemoglobin with the over-all alteration or damage to blood which may occur during extracorporeal perfusion. However, it is customary in most Centers where "open-heart" surgery is performed
Vol. 45, No. 2 February, 1963
HEMOLYSTS OF BLOOD
247
to rely upon this simple determination to measure relative degrees of blood " t r a u m a , " much as in Public Health Studies in which the "bacillus coli" count is used to indicate the degree of pollution of water. Canine and human blood do not respond to mechanical and chemical stress in the same way or, at least, not to the same degree. Canine blood appears to be more "fragile" than human blood, and thus might serve as a more sensitive indicator of the effects of "chemical" or other stress. To reduce the significance of this species difference, a smaller series of studies using fresh heparinized human blood has been carried out and is included in this report. METHOD
Three types of commercially available plastic tubing were chosen and were designated as types A,* B,f and C t Various sizes of tubing in common use for perfusion purposes were tested as indicated in the charts. mg. 1. 000
Fig. 2.—Plasma hemoglobin in plastic tubes (48 hr. after gas sterilization) incubated at 37° C. (canine blood). •Tyson S-22-1 (U. S. Stoneware Co., Akron, Ohio). tB-D Polyvinyl V-516 (Becton-Dickinson Co., Rutherford, N.J.). tMayon No. 0247 (Mayon Plastic, 17th Ave., North Hopkins, Minn.).
248
HIROSE, GOLDSTEIN, BAILEY
J. Thor.n-if and Cardiovas. Surf.
Fig. 3.—Plasma hemoglobin in plastic tubes (48 hr. after gas and steam sterilization) incubated at 37° C. (human blood).
Each type and size of tubing was cut into segments capable of containing 5 c.c. of blood. They were sterilized by steam for 20 minutes or by exposure to ethylene-oxide gas for 6 hours as indicated. The tubes were tested for their effect upon contained specimens of heparinized canine (or human) blood at periods of 1, 3, 5, and 7 days following sterilization. The canine blood was obtained by femoral cathetcrization, being collected in 500 c.e. quantities which were admixed with 50 nig. of hcparin sodium* in 50 c.c. of physiologic saline. The human blood was obtained in smaller quantities (but under sterile conditions and using similar concentrations of hcparin) from the arterial cannulae inserted into human patients just prior to performance of extracorporeal bypass procedures. Five cubic centimeter quantities of the sampled blood were introduced into each of the various tube segments, the ends of which then were clamped with ordinary hemostatie forceps. Control samples were collected in plain glass tubes. These glass and plastic tubes (with clamps) were incubated for various •The Upjohn Co., Kalamazoo. Mich.
Vol. 45, No. 2 February, 1965
HEMOLYSIS OF BLOOD
249
periods of time (2, 4, or 24 hours) at 37° C. At the end of the period of incubation the blood samples were poured into glass tubes and the red cells were removed by centrifugation. Free plasma hemoglobin was quantitated by the benzidiae method. Sampled bacteriologic cultures of these specimens consistently proved sterile. Each test was repeated several times and the results were averaged. RESULTS
Fig. 1 indicates the plasma hemoglobin values obtained from canine blood exposed to various sizes of the three types of plastic tubing .after 2 hours and 24 hours of incubation, respectively, the tubes having been sterilized with ethylenc-oxide gas 48 hours previously. Fig. 2 shows the effect on canine blood after 2, 4, and 24 hours of incubation, the tubes having been sterilized with ethylenc-oxide gas 48 hours previously. Fig. 3 shows the effect on human blood after 2, 4, and 24 hours of incubation, the tubes having been sterilized with ethylene-oxide gas or steam 48 hours previously. Fig. 4 shows the effect on
Fig. 4.—Effect of prolonged incubation upon canine blood in contact with plastic tubing sterilized by ethylene-oxide gas at various intervals before use.
HIROSE, GOLDSTEIN, BAILEY
J. Thoracic and Cardiovas. Surg.
mg. I, 000
W
Fig. 5.—Effect
—
"i
in
r-
y
i
^
t
n
m
i
^
( / ) _ , * >
m
p-
of prolonged incubation upon human blood in contact with plastic tubing sterilized by ethylene-oxide gas at various intervals before use.
canine blood at various times after sterilization of 24 hours of incubation. Pig. 5 shows the effect on human blood at various times after sterilization of 24 hours of incubation. DISCUSSION
In spite of the fact that this was a static experiment in that the blood was merely exposed to and not caused to circulate through these tubes, it is believed that some worth-while preliminary conclusions are possible. It is clearly evident that a certain amount of homolysis does occur merely from contact with plastic tubing of the types commonly used in cxtracorporeal circuits. This is appreciably greater after 24 hours of blood contact than after 2 hour periods of contact (at normothermic temperatures). Similar results were obtained in the respective canine and human blood experiments (actually Type B tubing showed better results in the human than in the canine blood experiments).
Vol. 45, No. 2 February, 1963
HEMOLYSIS OF BLOOD
95I ~ox
Unlike Keith and his associates,1 we did not observe any particular differences in the hemolytic effects of different batches of plastic material obtained from the same manufacturer. However, there were very great differences in the amounts of hemolysis produced by the various tested brands of plastic tubing. Several samples (especially Type A) necessarily were discarded from the study because of clot formation during the 24 hour incubation period. Although the same concentration of heparin sodium (50 mg. for 500 c.c. of blood) was used in each of these samples, none of the control samples in the glass tubes showed any clot formation. It appeared to us that this probably was related to destruction of heparin or absorption into the plastic tube walls during the 24 hour period of incubation. It was quite evident that recent exposure of the plastic material to ethyleneoxide gas produced changes in the tubing which induced greater degrees of hemolysis in the exposed blood. Whether this was due to the presence of some of the gas itself that had become absorbed into the tubing or whether it was related to temporary chemical or physical changes imparted to the plastic was not determined. In any event this effect was shown to be transient, being maximal immediately after sterilization and nearly absent (reduced to steam sterilization levels) when the exposure of the blood was made 7 days after sterilization. CONCLUSIONS
One of the commercially available types of plastic tubing (Type C) which was tested showed far less tendency to produce hemolysis in blood incubated in it than did either of the other two. Sterilization with ethylene-oxide gas temporarily exaggerated the hemolytic effects of all three types of tubing. It is concluded from these observations that at least 3 and, preferably, 5 days should be permitted to elapse before using gas-sterilized tubing for clinical extracorporeal bypass. While the increasing utilization of this method of sterilization of apparatus to be used for extracorporeal bypass is based upon the ' ' convenience'' of the method and the absence of gross physical alteration of the plastic components (no discoloration or change in flexibility), the demonstrated potential increase in blood damage associated with this sterilization technique may tend to restrict its popularity. REFERENCE 1. Keith, H. B., Ginn, B., Williams, G. K., and Campbell, G. S.:
Extracorporeal Circulation, .T. THORACIC SURG. 4 1 : 404, 1961.
Massive Hemolysis in
AND
STERILIZATION and
T
HE continuous improvements in the various kinds of equipment available for extracorporeal circulatory bypass and the development of more intricate operations for intracardiac pathologic conditions are reorienting our thoughts in the direction of longer and longer periods of perfusion. Partial circulatory assistance and hemodialysis imply even longer periods of bypass. The anticipated increase in time of exposure of the participate and fluid content of the blood to the various metallic or plastic components of the apparatus inevitably would seem to tend to exaggerate any hemolytic or other deleterious effects. It is generally admitted that such changes in the blood can precipitate serious visceral complications (such as renal or hepatic failure) even though the exact mechanisms of their effects may not be understood fully. Therefore, any contribution to the known methods of limiting or reducing such blood damage should be worth while. Since using the ethylene-oxide gas sterilization technique at the Flower and Fifth Avenue Hospitals in New York City for the preparation of our perfusion equipment, we have observed a significant increase in the incidence of renal complications. This has been associated with consistently higher reported levels of free hemoglobin in the perfused blood (using identical extracorporeal equipment for comparable periods of time). The question was raised whether ethylene-oxide gas might not be absorbed by or combined with the plastic materials used in the circuit, thus changing their nature and their effect on the blood. It was deemed improbable that this gas would combine with or be absorbed by the metallic (steel) or glass components of the equipment. The recent report of Keith and his associates1 concerning the enhanced hemolytic effect upon perfused blood of one batch of plastic tubing of a type commonly used for extracorporeal bypass suggested to From the New York Medical College, Flower and Fifth Avenue Hospitals, 1249 Fifth Ave., New York 29, N. Y. Aided by Grant H 6966 from the 17. S. Public Health Service and American Heart Association. Received for publication April 4, 1962. •Teaching- Fellow in Surgery. **Associate Professor of Medicine. "•Professor of Surgery. 245
HIROSE, GOLDSTEIN, B A I L E Y
246
J. Thoracic and Cardiovas. Suiy.
mg 1, 000
2 hours A
B
A
24 hours B
Pig. 1.—Plasma hemoglobin in different sizes of various plastic tubes (48 hr. after gas sterilization) incubated at 37° C. for 2 hr. and 24 hr. (canine blood).
us that different types and brands of the available plastic materials might have variable effects upon the blood and might be differentially affected by agents which could be absorbed by or might combine with them. Hence, it was decided to carry out a study of the effect upon sterile canine and human blood of exposure for periods up to 24 hours to various types (brands) and sizes of commonly used plastic tubing which had been sterilized, respectively, by steam or ethylene-oxide gas. It was realized at the outset that the contemplated studies would not be directly and fully referable to clinical problems of perfusion since the clinical and experimental conditions are not exactly comparable. In the present study no movement of the exposed blood has been incorporated in the test. It is intended that a later study will be oriented in the direction of evaluating damage due to movement of the blood through the several types of bypass equipment. Furthermore it is obvious that one cannot equate the level of plasma hemoglobin with the over-all alteration or damage to blood which may occur during extracorporeal perfusion. However, it is customary in most Centers where "open-heart" surgery is performed
Vol. 45, No. 2 February, 1963
HEMOLYSTS OF BLOOD
247
to rely upon this simple determination to measure relative degrees of blood " t r a u m a , " much as in Public Health Studies in which the "bacillus coli" count is used to indicate the degree of pollution of water. Canine and human blood do not respond to mechanical and chemical stress in the same way or, at least, not to the same degree. Canine blood appears to be more "fragile" than human blood, and thus might serve as a more sensitive indicator of the effects of "chemical" or other stress. To reduce the significance of this species difference, a smaller series of studies using fresh heparinized human blood has been carried out and is included in this report. METHOD
Three types of commercially available plastic tubing were chosen and were designated as types A,* B,f and C t Various sizes of tubing in common use for perfusion purposes were tested as indicated in the charts. mg. 1. 000
Fig. 2.—Plasma hemoglobin in plastic tubes (48 hr. after gas sterilization) incubated at 37° C. (canine blood). •Tyson S-22-1 (U. S. Stoneware Co., Akron, Ohio). tB-D Polyvinyl V-516 (Becton-Dickinson Co., Rutherford, N.J.). tMayon No. 0247 (Mayon Plastic, 17th Ave., North Hopkins, Minn.).
248
HIROSE, GOLDSTEIN, BAILEY
J. Thor.n-if and Cardiovas. Surf.
Fig. 3.—Plasma hemoglobin in plastic tubes (48 hr. after gas and steam sterilization) incubated at 37° C. (human blood).
Each type and size of tubing was cut into segments capable of containing 5 c.c. of blood. They were sterilized by steam for 20 minutes or by exposure to ethylene-oxide gas for 6 hours as indicated. The tubes were tested for their effect upon contained specimens of heparinized canine (or human) blood at periods of 1, 3, 5, and 7 days following sterilization. The canine blood was obtained by femoral cathetcrization, being collected in 500 c.e. quantities which were admixed with 50 nig. of hcparin sodium* in 50 c.c. of physiologic saline. The human blood was obtained in smaller quantities (but under sterile conditions and using similar concentrations of hcparin) from the arterial cannulae inserted into human patients just prior to performance of extracorporeal bypass procedures. Five cubic centimeter quantities of the sampled blood were introduced into each of the various tube segments, the ends of which then were clamped with ordinary hemostatie forceps. Control samples were collected in plain glass tubes. These glass and plastic tubes (with clamps) were incubated for various •The Upjohn Co., Kalamazoo. Mich.
Vol. 45, No. 2 February, 1965
HEMOLYSIS OF BLOOD
249
periods of time (2, 4, or 24 hours) at 37° C. At the end of the period of incubation the blood samples were poured into glass tubes and the red cells were removed by centrifugation. Free plasma hemoglobin was quantitated by the benzidiae method. Sampled bacteriologic cultures of these specimens consistently proved sterile. Each test was repeated several times and the results were averaged. RESULTS
Fig. 1 indicates the plasma hemoglobin values obtained from canine blood exposed to various sizes of the three types of plastic tubing .after 2 hours and 24 hours of incubation, respectively, the tubes having been sterilized with ethylenc-oxide gas 48 hours previously. Fig. 2 shows the effect on canine blood after 2, 4, and 24 hours of incubation, the tubes having been sterilized with ethylenc-oxide gas 48 hours previously. Fig. 3 shows the effect on human blood after 2, 4, and 24 hours of incubation, the tubes having been sterilized with ethylene-oxide gas or steam 48 hours previously. Fig. 4 shows the effect on
Fig. 4.—Effect of prolonged incubation upon canine blood in contact with plastic tubing sterilized by ethylene-oxide gas at various intervals before use.
HIROSE, GOLDSTEIN, BAILEY
J. Thoracic and Cardiovas. Surg.
mg. I, 000
W
Fig. 5.—Effect
—
"i
in
r-
y
i
^
t
n
m
i
^
( / ) _ , * >
m
p-
of prolonged incubation upon human blood in contact with plastic tubing sterilized by ethylene-oxide gas at various intervals before use.
canine blood at various times after sterilization of 24 hours of incubation. Pig. 5 shows the effect on human blood at various times after sterilization of 24 hours of incubation. DISCUSSION
In spite of the fact that this was a static experiment in that the blood was merely exposed to and not caused to circulate through these tubes, it is believed that some worth-while preliminary conclusions are possible. It is clearly evident that a certain amount of homolysis does occur merely from contact with plastic tubing of the types commonly used in cxtracorporeal circuits. This is appreciably greater after 24 hours of blood contact than after 2 hour periods of contact (at normothermic temperatures). Similar results were obtained in the respective canine and human blood experiments (actually Type B tubing showed better results in the human than in the canine blood experiments).
Vol. 45, No. 2 February, 1963
HEMOLYSIS OF BLOOD
95I ~ox
Unlike Keith and his associates,1 we did not observe any particular differences in the hemolytic effects of different batches of plastic material obtained from the same manufacturer. However, there were very great differences in the amounts of hemolysis produced by the various tested brands of plastic tubing. Several samples (especially Type A) necessarily were discarded from the study because of clot formation during the 24 hour incubation period. Although the same concentration of heparin sodium (50 mg. for 500 c.c. of blood) was used in each of these samples, none of the control samples in the glass tubes showed any clot formation. It appeared to us that this probably was related to destruction of heparin or absorption into the plastic tube walls during the 24 hour period of incubation. It was quite evident that recent exposure of the plastic material to ethyleneoxide gas produced changes in the tubing which induced greater degrees of hemolysis in the exposed blood. Whether this was due to the presence of some of the gas itself that had become absorbed into the tubing or whether it was related to temporary chemical or physical changes imparted to the plastic was not determined. In any event this effect was shown to be transient, being maximal immediately after sterilization and nearly absent (reduced to steam sterilization levels) when the exposure of the blood was made 7 days after sterilization. CONCLUSIONS
One of the commercially available types of plastic tubing (Type C) which was tested showed far less tendency to produce hemolysis in blood incubated in it than did either of the other two. Sterilization with ethylene-oxide gas temporarily exaggerated the hemolytic effects of all three types of tubing. It is concluded from these observations that at least 3 and, preferably, 5 days should be permitted to elapse before using gas-sterilized tubing for clinical extracorporeal bypass. While the increasing utilization of this method of sterilization of apparatus to be used for extracorporeal bypass is based upon the ' ' convenience'' of the method and the absence of gross physical alteration of the plastic components (no discoloration or change in flexibility), the demonstrated potential increase in blood damage associated with this sterilization technique may tend to restrict its popularity. REFERENCE 1. Keith, H. B., Ginn, B., Williams, G. K., and Campbell, G. S.:
Extracorporeal Circulation, .T. THORACIC SURG. 4 1 : 404, 1961.
Massive Hemolysis in