- Email: [email protected]
Fate of indium 111-labeled platelets during cardiopulmonary bypass performed with membrane and bubble oxygenators
J THoRAc
CARDIOVASC SURG
84:39-43, 1982
Fate of indium Ill-labeled platelets during cardiopulmonary bypass performed with membrane and bubble oxygenators To elucidate the effects of bubble and membrane oxygenators on platelet integrity, we developed a quantitative method of determining platelet lysis during cardiopulmonary bypass. Two groups of dogs whose platelets had been labeled with indium III were subjected to I hour of cardiopulmonary bypass. In Group A (bubble oxygenator), platelet lysis as measured by free plasma lllln levels increased from 6% ± 1% to 33% ± 7% during bypass. In Group B (membrane oxygenator), plasma IlIln levels increased from 5% ± 2% to 10% ± 6% during bypass (p < 0.01). After I hour of bypass, the ratio of IlIln-labeled platelets to prebypass levels was 36% ± 8% in Group A and 67% ± 9% in Group B. Platelet deposition on the oxygenator was greater in bubble oxygenators (19% ± 4% of total injected 1111n) than in membrane oxygenators (12% ± 3% (if total injected I I 'In]. These data indicate that membrane oxygenators maintain a higher circulating platelet count both intraoperatively and postoperatively and result in less platelet destruction than bubble oxygenators following I hour of cardiopulmonary bypass in dogs.
Kevin A. Peterson, M.D., Mrinal K. Dewanjee , Ph.D., and Michael P. Kaye, M.D., Rochester, Minn.
h e question of potential benefit of membrane oxygenation for short-term cardiopulmonary bypass is unsettled. Superiority of the membrane oxygenator in perfusions of longer than two hours has been suggested. I, 2 Although on theoretical grounds the absence of a bloodgas interface should result in less blood trauma, 3.4 it has been difficult to substantiate this hypothesis meaningfully during short perfusions. l . 5. 6 Use of cardiotomy suction and lack of sensitivity of some hematologic and biochemical studies have been proposed as reasons for failure to obtain data supporting the hypothesis. To elucidate a small but significant facet ofthis problem, we chose to compare the effects of membrane and bubble oxygenation on one component of blood, the platelet. We developed a sensitive, quantitative method of determining platelet lysis during cardiopulmonary bypass. Usingindium I I I-labeled platelets, we have been able to determine accurately the effects of both types of oxy-
genator on platelet lysis as well as circulating platelet concentration and entrapment of platelets within the oxygenator. The data obtained from these studies indicate a platelet sparing effect of the membrane oxygenator. Materials and methods
Addressfor reprints: Michael P. Kaye, M.D., Mayo Clinic, Rochester, Minn. 55905.
Groups. Fourteen mongrel dogs weighing from 15 to 20 kg underwent cardiopulmonary bypass for 1 hour. In seven dogs (Group A), bypass was established with a bubble oxygenator (BOS-5, Bentley Laboratories, Inc., Irvine, Calif.) primed with 1,000 ml of electrolyte (Plasma-Lyte) solution. A Sarns bubble trap was placed in the arterial line (Sarns, Inc., Ann Arbor, Mich.). In the other seven dogs (Group B), bypass was established with a membrane oxygenator (3500-2A, SciMed Life Systems, Inc., Minneapolis, Minn.) primed with 1,500 ml of the same electrolyte solution. The membrane oxygenator was primed as follows: The circuit was flushed for 10 minutes with carbon dioxide, evacuated with a vacuum, primed, and the priming fluid was recirculated to remove bubbles. In all other ways the two groups were treated identically. Labeling. 111 In-labeled 8-hydroxyquinoline was prepared by mixing 400 to 600 /LCi of IIIIn-CI 3 (Medi-
© \982 The C. V. Mosby Co.
39
From the Mayo Clinic and Foundation, Rochester, Minn. Received for publication May 12, \981. Accepted for publication Sept. 28, 1981.
0022-5223/82/070039+05$00.50/0
The Journal of Thoracic and Cardiovascular Surgery
40 Peterson, Dewanjee, Kaye
bypass on
I-
bypass
-,
off
50 40
%111 In in
Plasma
30
III In in
Blood
20 10
o 10 20 30 40 50 60
120
180
240
Time (minutes) Fig. 1. Platelet destruction in Group A (bubble oxygenators, 0---0) and Group B (membrane oxygenators, _ ) prior to, during, and following cardiopulmonary bypass. Physics, Inc., Emeryville, Calif.) with 50 J-Lg of oxine in 50 ml of alcohol. On the day prior to operation, 47 ml of blood was withdrawn from each experimental animal, and the platelets were removed and labeled according to the method of Heaton and 'associates" as modified by Dewanjee and colleagues. 8 Using this technique, we have obtained labeling efficiencies of 65% to 80%. The labeled platelets were washed, suspended in 5 ml of acid-citrate-dextrose solution, and reinjected within 3 hours of the time of collection. Experimental design. Sixteen to 20 hours after injection of IIIIn-labeled platelets, the dogs were anesthetized with intravenous pentobarbital, intubated, and placed on a respirator. Next, 30 J-LCi of 125I-labeled human serum albumin was administered intravenously at least 15 minutes prior to operation to quantitate hemodilution. The femoral artery was exposed, a thoracotomy performed through the fourth left intercostal space, and the pericardium opened. Following systemic heparinization (3 mg/kg), the femoral artery was cannulated and a single venous cannula was placed in the right atrium. Cardiopulmonary bypass was established with the use of either a bubble oxygenator or a membrane oxygenator with silicone rubber tubing and a nonocclusive Mayo-Gibbons pump without arterial line filters. Dogs were subjected to cardiopulmonary bypass for I hour, including systemic hypothermia to 25° C for 30 minutes. At the completion of bypass, all blood in the oxygenator was returned to the animal. No cardiotomy suction was used, but the left ventricle was
vented through the left atrial appendage in all animals. At the completion of bypass, the oxygenator was removed from the circuit and gently rinsed with 1,000 ml of electrolyte solution. Following this, the oxygenator was imaged at 174 to 247 keV with a gamma camera (PG-V, Searle Radiographies, Inc., Des Plaines, Ill.). The core polyurethane mesh in the bubble oxygenator and the silicone rubber spiral coil in the membrane oxygenator were removed and measured for HlIn content in an ionization chamber (5R, Capintec, Inc., Montvale, N. J.). Tubing, reservoir bags, plastic, and other parts of the oxygenators were sampled and found to contain very little IHIn when evaluated in the ionization chamber. To determine platelet concentration and degree of lysis, we removed a 5 ml sample of blood from the left jugular venous line prior to the operation and at 5, 10, 20, 30, 60, 120, 180, and 240 minutes after initiation of bypass. A 1.2 ml aliquot was removed from each sample and centrifuged at 25,000 g for 10 minutes. The plasma supernatant was removed from the centrifuged aliquots, and the fractions and whole blood samples were measured for IHIn and 1251 with a gamma well counter (Beckman Gamma 8000, Beckman Instruments, Inc., Fullerton, Calif.) adjusted to incorporate the 174,247, and 421 keV sum peaks and the 28 to 35 keV peaks of 1251 with a crossover correction for IIIIn. Values obtained for the plasma-free fraction represent platelet-bound HlJn. Mean values with standard deviations were calculated for IHIn in the oxygenator,
Volume 84
Platelet lysis during CPR
Number 1 July, 1982
bypass on
II
100
4 I
bypass off
II
r---------------------------.......
80
% III In-platelets
Platelets (prebypass)
60 40
20
o
10 20 30 40 50 60
120
160
240
Time (minutes) Fig. 2. Dynamic circulating platelet concentration in Group A (bubble oxygenators, 0 - 0 ) and Group B (membrane oxygenators, e_ _) prior to, during, and following cardiopulmonary bypass.
plasma, and whole blood. Platelet lysis was determined by comparing individual centrifuged fractions; in this manner, the problems caused by dilution inequality between groups were avoided. 1251-labeled human serum albumin concentrations were calculated and platelet 11lln values were corrected for hemodilution expected from 1,000 ml (bubble) or 1,500 ml (membrane) of clear oxygenator prime. Significant difference between paired sample data in Group A and Group B was determined by the Wilcoxon, Mann, and Whitney rank sum tests. Results Platelet destruction. In Group A (bubble oxygenator), platelet-bound 111ln was released into plasma throughout bypass. Plasma 111ln levels increased from 6% ± 1% (SD) of blood lllln levels before bypass to 33% ± 7% of blood 1IIIn levels after I hour of bypass (Fig. 1). Following completion of the bypass procedure, plasma lllln declined, most likely because of redistribution and excretion. In contrast, plasma IllIn levels in Group B (membrane oxygenator) increased from 5% ± 2% of blood lllln levels before bypass to 10% ± 6% of blood 111ln levels after 10 minutes of bypass. Thereafter the plasma 111ln levels remained constant, there being no additional platelet breakdown during the remainder of bypass. The difference in values for plasma lllln levels at the completion of bypass was significant at the p < 0.01 level. Platelet concentration and hemodilution. Although
some 1251-labeled albumin is absorbed onto the extracorporeal circuit in a time-dependent fashion," the total absorbed albumin fraction is small. As a stable protein, albumin dilution therefore accurately reflects dilution of blood elements due to oxygenator prime. In Group A, 1251 declined' to a mean of 66% ± 7% of initial concentration with 5 minutes of bypass and remained steady. After 4 hours, the mean was 73% ± 8%. 1251 levels for membrane oxygenators dropped to a mean of 45% ± 14% of initial concentration and also remained steady. After 4 hours, the mean was 58% ± 9% of initial concentration. These values approximate those calculated by assuming 8% of body weight as circulating blood volume and the difference in the volume of prime in each group. Dynamic circulating platelets, as measured by platelet-bound lllIn, declined in Group A within 5 minutes after initiation of bypass to 61 % ± 11% of initial platelet levels, a value approximating the dilution values (Fig. 2). In this group, platelet-bound lllln continued to decline and, after 20 minutes, reached a minimum of 29% ± 6% of initial platelet levels. In Group A this value remained low, being 36% ± 8% after 1 hour of bypass. Following the bypass procedure, platelet concentration increased slowly over 3 hours to 51 % ± 10% of initial levels. In Group B, there was an initial decline over 10 minutes following the initiation of bypass to 38% ± 19% of initial circulating platelet levels (again a value approximating dilutional values). Platelet-bound lllln
42 Peterson, Dewanjee, Kaye
then increased steadily to 67% ± 9% after 1 hour of cardiopulmonary bypass. Following completion of the bypass procedure, platelet concentration continued to increase and 3 hours postoperatively was 92% ± 8% of initial platelet concentration. Oxygenator deposition. Platelet deposition in the extracorporeal circuit occurred almost entirely in the polyurethane mesh of the bubble oxygenator and the spiral coil membrane of the membrane oxygenator. Tubing, reservoir bags, plastic, and other parts of the oxygenators were found to contain very little radioactivity and were not included in calculations of oxygenator IIlIn content. Gamma camera scintiphotographs demonstrated platelet deposition on both types of oxygenators. In Group A, the polyurethane mesh removed from the core of the bubble oxygenator was found to contain 19% ± 4% of total injected IIlIn of the day before (corrected for decay). The silicone rubber spiral coil membrane removed from the membrane oxygenator of Group B contained 12% ± 3% of total injected IIIIn of the day before.
Discussion Deficits of platelet number and function may be an important cause of impaired hemostasis following cardiopulmonary bypass. 10. II These deficits are presumably produced after platelet interaction with the oxygenator in the extracorporeal circuit. 3, 6 Membrane oxygenators have been demonstrated to result in higher intraoperative platelet levels than bubble oxygenators during procedures of greater than 2 hours. I, 2, 12 However, the effect of membrane oxygenators on platelets during shorter cardiopulmonary perfusion time has not been significantly different from the effect of bubble oxygenators, 4, 7 With the introduction of IIIIn labeling techniques ,9 a new tool has been made available for the investigation of platelet kinetics and deposition. Since no significant amount of IllIn label is released from the platelet during activation and degranulation.P the platelet IIlIn level remains separate from the blood plasma fraction until destruction of the platelets occurs. The IIIIn label in platelets is bound to a platelet cytoplasmic protein of molecular weight 50,000. 14 Since rupture of the platelet membrane results in loss of this protein into the plasma, platelet breakdown may be quantified by measuring serial plasma IllIn levels. This characteristic, along with the high yield of IIIIn platelets and efficient gammaemitting properties of IIIIn, allows for the sensitive examination of platelet response during cardiopulmonary bypass.
The Journal of Thoracic and Cardiovascular Surgery
Our study demonstrates an increase in plasma IIIIn, indicating continued platelet destruction, throughout cardiopulmonary bypass when bubble oxygenators are used. Platelet destruction is significantly reduced when membrane oxygenators are used, with platelet destruction occurring only within the first 10 minutes of bypass, During cardiopulmonary bypass with membrane oxygenators, platelet concentration remains higher intraoperatively and become higher postoperatively than when bubble oxygenators are used. Our findings are consistent with those of Addonizio and co-workers, 15 who demonstrated maximum platelet loss in membrane oxygenators to occur very early following initiation of bypass. In our study, a large decrease in platelet concentration occured within 10 minutes after initiation of bypass, and this decrease cannot be attributed to hemodilution alone. The loss of dynamically circulating platelets is likely the result ofrapid initial platelet adherence to the oxygenator surface or platelet sequestration by the liver and spleen, 2 The thesis of surface adherence tends to be supported by our demonstration that 19% of the total injected platelets are deposited on bubble oxygenators and 12% of the total injected platelets are deposited on membrane oxygenators. Although 1251_ labeled albumin remains diluted, platelet concentration appears to increase over 3 hours postoperatively. The cause of this increasing platelet concentration remains unclear; however, possible explanations include decreasing effects of barbiturate anesthesia with splenic contraction or, possibly, a compensatory redistribution of platelets from organs, vessels, and bone marrow, The mechanism of platelet destruction during cardiopulmonary bypass remains unclear, One probable mechanism is platelet membrane damage by shearing forces, either by contact with foreign surfaces with partial release of platelet content or by contact with the blood-gas interface in the bubble oxygenator.!" Membrane oxygenators may reduce platelet destruction by allowing oxygen to diffuse through a semipermeable membrane and thus prevent the blood-gas interface. Using IIIIn labeling techniques, we have demonstrated that the membrane oxygenators cause less platelet destruction than bubble oxygenators following I hour of cardiopulmonary bypass in dogs, We are grateful for the technical assistance of Mr. Arlan Hildestad, Miss Rebecca Pluth, and Mr. John Ritter.
REFERENCES Clark RE, Beauchamp RA, Magrath RA, Brooks 10, Ferguson TB, Weldon CS: Comparison of bubble and
Volume 84
Platelet lysis during CPR
Number 1
43
July, 1982
membrane oxygenators in short and long perfusions. J THORAC CARDIOVASC SURG 78:655-666, 1979 2 de Leval M, Hill JD, Mielke CH Jr, Macur MF, Gerbode F: Blood platelets and extracorporeal circulation. Kinetic studies on dogs on cardiopulmonary bypass. J THoRAc CARDIOVASC SURG 69:144-151,1975 3 Peirce EC III: Is the blood-gas interface of clinical importance? Ann Thorac Surg 17:526-529, 1974 4 Siderys H, Herod GT, Holbrook H, Pittman IN, Rubush JL, Kasebaker V, Berry GR Jr: Membrane and bubble oxygenation as used in cardiopulmonary bypass in patients. J THoRAc CARDIOVASC SURG 69:708-712, 1974 5 Williams DR, Tyers GFO, Williams EH, Kurusz M, Shaffer CW, Pierce WS, Waldhausen JA: Similarity of clinical and laboratory results obtained with microporous Teflon membrane oxygenator and bubble-film hybrid oxygenator. Ann Thorac Surg 25:30-35, 1978 6 Friedenberg WR, Myers WO, Plotka ED, Beathard IN, Kummer DJ, Gatlin PF, Stoiber DL, Ray JF III, Sautter RD: Platelet dysfunction associated with cardiopulmonary bypass. Ann Thorac Surg 25:298-305, 1978 7 Heaton WA, Davis HH, Welch MJ, Mathias CJ, Joist JH, Sherman LA, Siegel BA: Indium-III: A new radionuelide label for studying human platelet kinetics. Br J Haematol 42:613-622, 1979 8 Dewanjee MK, Fuster V, Kaye MP, Josa M: Imaging platelet deposition with II 'In-labeled platelets in coronary artery bypass grafts in dogs. Mayo Clin Proc 53:327-331, 1978 9 Addonizio VP Jr, Macarak EJ, Nicolaou KC, Edmunds
10
II
12
13
14
15
LH Jr, Colman RW: Effects of prostacyclin and albumin on platelet loss during in vitro simulation of extracorporeal circulation. Blood 53: 1033-1042, 1979 Bick RL: Alterations of hemostasis associated with cardiopulmonary bypass. Pathophysiology, prevention, diagnosis, and management, Seminars in Thrombosis and Hemostasis, EF Mammen, ed., New York, 1976, Stratton Intercontinental, Medical Book Corp., Publisher, p 59 Andersen MN, Hambraeus G: Physiologic and biochemical responses to prolonged extracorporeal circulation. Experimental studies during four-hour perfusion. Ann Surg 153:592-598, 1961 Addonizio VP Jr, Smith JB, Strauss JF III, Colman RW, Edmunds LH Jr: Thromboxane synthesis and platelet secretion during cardiopulmonary bypass with bubble oxygenator. J THoRAc CARDIOVASC SURG 79:91-96, 1980 Joist JH, Baker RK, Thakur ML, Welch MJ: IndiumIII-labeled human platelets. Uptake and loss of label and in vitro function of labeled platelets. J Lab Clin Med 92:829-836, 1978 Hudson EM, Ramsey RB, Evatt BL: Localization of 111indium in subcellular components of lliindium labeled human platelets. Proc Joint Meeting 18th Cong Int Soc Hematol and 16th Cong Int Soc Blood Transfusion 225, 1980 Addonizio VP Jr, Macarak EJ, Niewiarowski S, Colman RW, Edmunds LH Jr: Preservation of human platelets with prostaglandin E) during in vitro simulation of cardiopulmonary bypass. Circ Res 44:350-357, 1979
CARDIOVASC SURG
84:39-43, 1982
Fate of indium Ill-labeled platelets during cardiopulmonary bypass performed with membrane and bubble oxygenators To elucidate the effects of bubble and membrane oxygenators on platelet integrity, we developed a quantitative method of determining platelet lysis during cardiopulmonary bypass. Two groups of dogs whose platelets had been labeled with indium III were subjected to I hour of cardiopulmonary bypass. In Group A (bubble oxygenator), platelet lysis as measured by free plasma lllln levels increased from 6% ± 1% to 33% ± 7% during bypass. In Group B (membrane oxygenator), plasma IlIln levels increased from 5% ± 2% to 10% ± 6% during bypass (p < 0.01). After I hour of bypass, the ratio of IlIln-labeled platelets to prebypass levels was 36% ± 8% in Group A and 67% ± 9% in Group B. Platelet deposition on the oxygenator was greater in bubble oxygenators (19% ± 4% of total injected 1111n) than in membrane oxygenators (12% ± 3% (if total injected I I 'In]. These data indicate that membrane oxygenators maintain a higher circulating platelet count both intraoperatively and postoperatively and result in less platelet destruction than bubble oxygenators following I hour of cardiopulmonary bypass in dogs.
Kevin A. Peterson, M.D., Mrinal K. Dewanjee , Ph.D., and Michael P. Kaye, M.D., Rochester, Minn.
h e question of potential benefit of membrane oxygenation for short-term cardiopulmonary bypass is unsettled. Superiority of the membrane oxygenator in perfusions of longer than two hours has been suggested. I, 2 Although on theoretical grounds the absence of a bloodgas interface should result in less blood trauma, 3.4 it has been difficult to substantiate this hypothesis meaningfully during short perfusions. l . 5. 6 Use of cardiotomy suction and lack of sensitivity of some hematologic and biochemical studies have been proposed as reasons for failure to obtain data supporting the hypothesis. To elucidate a small but significant facet ofthis problem, we chose to compare the effects of membrane and bubble oxygenation on one component of blood, the platelet. We developed a sensitive, quantitative method of determining platelet lysis during cardiopulmonary bypass. Usingindium I I I-labeled platelets, we have been able to determine accurately the effects of both types of oxy-
genator on platelet lysis as well as circulating platelet concentration and entrapment of platelets within the oxygenator. The data obtained from these studies indicate a platelet sparing effect of the membrane oxygenator. Materials and methods
Addressfor reprints: Michael P. Kaye, M.D., Mayo Clinic, Rochester, Minn. 55905.
Groups. Fourteen mongrel dogs weighing from 15 to 20 kg underwent cardiopulmonary bypass for 1 hour. In seven dogs (Group A), bypass was established with a bubble oxygenator (BOS-5, Bentley Laboratories, Inc., Irvine, Calif.) primed with 1,000 ml of electrolyte (Plasma-Lyte) solution. A Sarns bubble trap was placed in the arterial line (Sarns, Inc., Ann Arbor, Mich.). In the other seven dogs (Group B), bypass was established with a membrane oxygenator (3500-2A, SciMed Life Systems, Inc., Minneapolis, Minn.) primed with 1,500 ml of the same electrolyte solution. The membrane oxygenator was primed as follows: The circuit was flushed for 10 minutes with carbon dioxide, evacuated with a vacuum, primed, and the priming fluid was recirculated to remove bubbles. In all other ways the two groups were treated identically. Labeling. 111 In-labeled 8-hydroxyquinoline was prepared by mixing 400 to 600 /LCi of IIIIn-CI 3 (Medi-
© \982 The C. V. Mosby Co.
39
From the Mayo Clinic and Foundation, Rochester, Minn. Received for publication May 12, \981. Accepted for publication Sept. 28, 1981.
0022-5223/82/070039+05$00.50/0
The Journal of Thoracic and Cardiovascular Surgery
40 Peterson, Dewanjee, Kaye
bypass on
I-
bypass
-,
off
50 40
%111 In in
Plasma
30
III In in
Blood
20 10
o 10 20 30 40 50 60
120
180
240
Time (minutes) Fig. 1. Platelet destruction in Group A (bubble oxygenators, 0---0) and Group B (membrane oxygenators, _ ) prior to, during, and following cardiopulmonary bypass. Physics, Inc., Emeryville, Calif.) with 50 J-Lg of oxine in 50 ml of alcohol. On the day prior to operation, 47 ml of blood was withdrawn from each experimental animal, and the platelets were removed and labeled according to the method of Heaton and 'associates" as modified by Dewanjee and colleagues. 8 Using this technique, we have obtained labeling efficiencies of 65% to 80%. The labeled platelets were washed, suspended in 5 ml of acid-citrate-dextrose solution, and reinjected within 3 hours of the time of collection. Experimental design. Sixteen to 20 hours after injection of IIIIn-labeled platelets, the dogs were anesthetized with intravenous pentobarbital, intubated, and placed on a respirator. Next, 30 J-LCi of 125I-labeled human serum albumin was administered intravenously at least 15 minutes prior to operation to quantitate hemodilution. The femoral artery was exposed, a thoracotomy performed through the fourth left intercostal space, and the pericardium opened. Following systemic heparinization (3 mg/kg), the femoral artery was cannulated and a single venous cannula was placed in the right atrium. Cardiopulmonary bypass was established with the use of either a bubble oxygenator or a membrane oxygenator with silicone rubber tubing and a nonocclusive Mayo-Gibbons pump without arterial line filters. Dogs were subjected to cardiopulmonary bypass for I hour, including systemic hypothermia to 25° C for 30 minutes. At the completion of bypass, all blood in the oxygenator was returned to the animal. No cardiotomy suction was used, but the left ventricle was
vented through the left atrial appendage in all animals. At the completion of bypass, the oxygenator was removed from the circuit and gently rinsed with 1,000 ml of electrolyte solution. Following this, the oxygenator was imaged at 174 to 247 keV with a gamma camera (PG-V, Searle Radiographies, Inc., Des Plaines, Ill.). The core polyurethane mesh in the bubble oxygenator and the silicone rubber spiral coil in the membrane oxygenator were removed and measured for HlIn content in an ionization chamber (5R, Capintec, Inc., Montvale, N. J.). Tubing, reservoir bags, plastic, and other parts of the oxygenators were sampled and found to contain very little IHIn when evaluated in the ionization chamber. To determine platelet concentration and degree of lysis, we removed a 5 ml sample of blood from the left jugular venous line prior to the operation and at 5, 10, 20, 30, 60, 120, 180, and 240 minutes after initiation of bypass. A 1.2 ml aliquot was removed from each sample and centrifuged at 25,000 g for 10 minutes. The plasma supernatant was removed from the centrifuged aliquots, and the fractions and whole blood samples were measured for IHIn and 1251 with a gamma well counter (Beckman Gamma 8000, Beckman Instruments, Inc., Fullerton, Calif.) adjusted to incorporate the 174,247, and 421 keV sum peaks and the 28 to 35 keV peaks of 1251 with a crossover correction for IIIIn. Values obtained for the plasma-free fraction represent platelet-bound HlJn. Mean values with standard deviations were calculated for IHIn in the oxygenator,
Volume 84
Platelet lysis during CPR
Number 1 July, 1982
bypass on
II
100
4 I
bypass off
II
r---------------------------.......
80
% III In-platelets
Platelets (prebypass)
60 40
20
o
10 20 30 40 50 60
120
160
240
Time (minutes) Fig. 2. Dynamic circulating platelet concentration in Group A (bubble oxygenators, 0 - 0 ) and Group B (membrane oxygenators, e_ _) prior to, during, and following cardiopulmonary bypass.
plasma, and whole blood. Platelet lysis was determined by comparing individual centrifuged fractions; in this manner, the problems caused by dilution inequality between groups were avoided. 1251-labeled human serum albumin concentrations were calculated and platelet 11lln values were corrected for hemodilution expected from 1,000 ml (bubble) or 1,500 ml (membrane) of clear oxygenator prime. Significant difference between paired sample data in Group A and Group B was determined by the Wilcoxon, Mann, and Whitney rank sum tests. Results Platelet destruction. In Group A (bubble oxygenator), platelet-bound 111ln was released into plasma throughout bypass. Plasma 111ln levels increased from 6% ± 1% (SD) of blood lllln levels before bypass to 33% ± 7% of blood 1IIIn levels after I hour of bypass (Fig. 1). Following completion of the bypass procedure, plasma lllln declined, most likely because of redistribution and excretion. In contrast, plasma IllIn levels in Group B (membrane oxygenator) increased from 5% ± 2% of blood lllln levels before bypass to 10% ± 6% of blood 111ln levels after 10 minutes of bypass. Thereafter the plasma 111ln levels remained constant, there being no additional platelet breakdown during the remainder of bypass. The difference in values for plasma lllln levels at the completion of bypass was significant at the p < 0.01 level. Platelet concentration and hemodilution. Although
some 1251-labeled albumin is absorbed onto the extracorporeal circuit in a time-dependent fashion," the total absorbed albumin fraction is small. As a stable protein, albumin dilution therefore accurately reflects dilution of blood elements due to oxygenator prime. In Group A, 1251 declined' to a mean of 66% ± 7% of initial concentration with 5 minutes of bypass and remained steady. After 4 hours, the mean was 73% ± 8%. 1251 levels for membrane oxygenators dropped to a mean of 45% ± 14% of initial concentration and also remained steady. After 4 hours, the mean was 58% ± 9% of initial concentration. These values approximate those calculated by assuming 8% of body weight as circulating blood volume and the difference in the volume of prime in each group. Dynamic circulating platelets, as measured by platelet-bound lllIn, declined in Group A within 5 minutes after initiation of bypass to 61 % ± 11% of initial platelet levels, a value approximating the dilution values (Fig. 2). In this group, platelet-bound lllln continued to decline and, after 20 minutes, reached a minimum of 29% ± 6% of initial platelet levels. In Group A this value remained low, being 36% ± 8% after 1 hour of bypass. Following the bypass procedure, platelet concentration increased slowly over 3 hours to 51 % ± 10% of initial levels. In Group B, there was an initial decline over 10 minutes following the initiation of bypass to 38% ± 19% of initial circulating platelet levels (again a value approximating dilutional values). Platelet-bound lllln
42 Peterson, Dewanjee, Kaye
then increased steadily to 67% ± 9% after 1 hour of cardiopulmonary bypass. Following completion of the bypass procedure, platelet concentration continued to increase and 3 hours postoperatively was 92% ± 8% of initial platelet concentration. Oxygenator deposition. Platelet deposition in the extracorporeal circuit occurred almost entirely in the polyurethane mesh of the bubble oxygenator and the spiral coil membrane of the membrane oxygenator. Tubing, reservoir bags, plastic, and other parts of the oxygenators were found to contain very little radioactivity and were not included in calculations of oxygenator IIlIn content. Gamma camera scintiphotographs demonstrated platelet deposition on both types of oxygenators. In Group A, the polyurethane mesh removed from the core of the bubble oxygenator was found to contain 19% ± 4% of total injected IIlIn of the day before (corrected for decay). The silicone rubber spiral coil membrane removed from the membrane oxygenator of Group B contained 12% ± 3% of total injected IIIIn of the day before.
Discussion Deficits of platelet number and function may be an important cause of impaired hemostasis following cardiopulmonary bypass. 10. II These deficits are presumably produced after platelet interaction with the oxygenator in the extracorporeal circuit. 3, 6 Membrane oxygenators have been demonstrated to result in higher intraoperative platelet levels than bubble oxygenators during procedures of greater than 2 hours. I, 2, 12 However, the effect of membrane oxygenators on platelets during shorter cardiopulmonary perfusion time has not been significantly different from the effect of bubble oxygenators, 4, 7 With the introduction of IIIIn labeling techniques ,9 a new tool has been made available for the investigation of platelet kinetics and deposition. Since no significant amount of IllIn label is released from the platelet during activation and degranulation.P the platelet IIlIn level remains separate from the blood plasma fraction until destruction of the platelets occurs. The IIIIn label in platelets is bound to a platelet cytoplasmic protein of molecular weight 50,000. 14 Since rupture of the platelet membrane results in loss of this protein into the plasma, platelet breakdown may be quantified by measuring serial plasma IllIn levels. This characteristic, along with the high yield of IIIIn platelets and efficient gammaemitting properties of IIIIn, allows for the sensitive examination of platelet response during cardiopulmonary bypass.
The Journal of Thoracic and Cardiovascular Surgery
Our study demonstrates an increase in plasma IIIIn, indicating continued platelet destruction, throughout cardiopulmonary bypass when bubble oxygenators are used. Platelet destruction is significantly reduced when membrane oxygenators are used, with platelet destruction occurring only within the first 10 minutes of bypass, During cardiopulmonary bypass with membrane oxygenators, platelet concentration remains higher intraoperatively and become higher postoperatively than when bubble oxygenators are used. Our findings are consistent with those of Addonizio and co-workers, 15 who demonstrated maximum platelet loss in membrane oxygenators to occur very early following initiation of bypass. In our study, a large decrease in platelet concentration occured within 10 minutes after initiation of bypass, and this decrease cannot be attributed to hemodilution alone. The loss of dynamically circulating platelets is likely the result ofrapid initial platelet adherence to the oxygenator surface or platelet sequestration by the liver and spleen, 2 The thesis of surface adherence tends to be supported by our demonstration that 19% of the total injected platelets are deposited on bubble oxygenators and 12% of the total injected platelets are deposited on membrane oxygenators. Although 1251_ labeled albumin remains diluted, platelet concentration appears to increase over 3 hours postoperatively. The cause of this increasing platelet concentration remains unclear; however, possible explanations include decreasing effects of barbiturate anesthesia with splenic contraction or, possibly, a compensatory redistribution of platelets from organs, vessels, and bone marrow, The mechanism of platelet destruction during cardiopulmonary bypass remains unclear, One probable mechanism is platelet membrane damage by shearing forces, either by contact with foreign surfaces with partial release of platelet content or by contact with the blood-gas interface in the bubble oxygenator.!" Membrane oxygenators may reduce platelet destruction by allowing oxygen to diffuse through a semipermeable membrane and thus prevent the blood-gas interface. Using IIIIn labeling techniques, we have demonstrated that the membrane oxygenators cause less platelet destruction than bubble oxygenators following I hour of cardiopulmonary bypass in dogs, We are grateful for the technical assistance of Mr. Arlan Hildestad, Miss Rebecca Pluth, and Mr. John Ritter.
REFERENCES Clark RE, Beauchamp RA, Magrath RA, Brooks 10, Ferguson TB, Weldon CS: Comparison of bubble and
Volume 84
Platelet lysis during CPR
Number 1
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LH Jr, Colman RW: Effects of prostacyclin and albumin on platelet loss during in vitro simulation of extracorporeal circulation. Blood 53: 1033-1042, 1979 Bick RL: Alterations of hemostasis associated with cardiopulmonary bypass. Pathophysiology, prevention, diagnosis, and management, Seminars in Thrombosis and Hemostasis, EF Mammen, ed., New York, 1976, Stratton Intercontinental, Medical Book Corp., Publisher, p 59 Andersen MN, Hambraeus G: Physiologic and biochemical responses to prolonged extracorporeal circulation. Experimental studies during four-hour perfusion. Ann Surg 153:592-598, 1961 Addonizio VP Jr, Smith JB, Strauss JF III, Colman RW, Edmunds LH Jr: Thromboxane synthesis and platelet secretion during cardiopulmonary bypass with bubble oxygenator. J THoRAc CARDIOVASC SURG 79:91-96, 1980 Joist JH, Baker RK, Thakur ML, Welch MJ: IndiumIII-labeled human platelets. Uptake and loss of label and in vitro function of labeled platelets. J Lab Clin Med 92:829-836, 1978 Hudson EM, Ramsey RB, Evatt BL: Localization of 111indium in subcellular components of lliindium labeled human platelets. Proc Joint Meeting 18th Cong Int Soc Hematol and 16th Cong Int Soc Blood Transfusion 225, 1980 Addonizio VP Jr, Macarak EJ, Niewiarowski S, Colman RW, Edmunds LH Jr: Preservation of human platelets with prostaglandin E) during in vitro simulation of cardiopulmonary bypass. Circ Res 44:350-357, 1979