Membrane oxygenation in baboons without anticoagulants

JOURNAL

OF SURGICAL

Membrane

RESEARCH

22,273-280

Oxygenation

J. RAYMOND

(1977)

in Baboons

without

FLETCHER, M.D. ,* ADAM E. MCKEE, CLIFFORD M. HERMAN, M.D.*

Anticoagulants1 D.V.M. ,t AND

Naval Medical Research Institute, *Experimental Surgery and Physiology Division and TDepartment of Experimental Pathology, Bethesda, Maryland 20014 Submitted for publication

November

23. 1976

Excessive bleeding continues to be a The present study was proposed to major clinical problem after perfusion for answer the following questions. (1) Is systemic heparinization necessary open heart surgery and during perfusion for long-term extracorporeal pulmonary sup- for 5 hr of venovenous membrane oxygenaport [I, 5, 7, 9, 10, 17, 18, 20-231. These tion in baboons? (2) Is systemic heparin superior to the use bleeding complications have been attributed to protein denaturation, metabolic acidosis, of no anticoagulant at all with respect to thrombocytopenia, hemostatic incompe- maintenance of arterial blood pressure, tence of the platelets and blood, undesired coagulation state, and scanning electron heparin effects, and excessive fibrinolysis microscopic appearance and function of the [I, 4, 7, 10, 221. The lack of methods membrane oxygenator? for precise control of the anticoagulant used (3) What are the differences between dog during perfusions has received recent atten- and baboon perfusions with and without tion as another unresolved problem area anticoagulants? [2, 141. Although bleeding during and MATERIALS AND METHODS after perfusions may be related to excessive heparin levels, the etiology is uncertain, Adult male baboons (20-30 kg) were and control with protamine and blood anesthetized with 1 mg/kg of Sernylan component therapy has not been completely (Parke, Davis), intubated, and allowed to satisfactory. breathe spontaneously. Through a right These difficulties led us to reexamine neck incision a single lumen (24 French) the entire approach to anticoagulation for polyvinyl chloride cannula was inserted via cardiopulmonary bypass. In our first study, the internal jugular vein to the level of dogs were successfully perfused for 24 hr the right atrium. A second cannula (22 without anticoagulants. The coagulation French) was positioned in the inferior changes seen during perfusion were similar vena cava via the left common femoral whether or not heparin was used. Dogs vein. A small cannula was inserted into the with no anticoagulants had better systemic femoral artery through a groin incision for arterial pressures and less deposition of pressure monitoring and blood sampling. A fibrin and formed elements on the surfaces rectal thermometer was used to determine of the circuits when compared to heparin- the animals’ temperature during the perfutreated animals [3]. sions. The baboons were then placed in chairs designed to allow maintenance of an ’ This study was supported by Clinical Investigation upright posture. Project S-06-459. The opinions or assertions contained The perfusion circuit consisted of sterile herein are the private ones of the authors and are not polyvinyl chloride tubing attached by polyto be construed as official or reflecting the views of carbonate connectors to a 1.5-m2 membrane the Navy Department or the Naval Service at large. 273 CopyrIght All right\

0 1977 by Academx Press. Inc. of reproducfion in any form reserved.

ISSN 0022-4804

274

JOURNAL

OF SURGICAL

RESEARCH:

oxygenator (Travenol, Modulung). An electromagnetic flow probe (Biotronex, BL615) was present on the venous inflow line of the oxygenator to determine the blood flow during the perfusion. Prelung pressure was measured by a transducer (Biotronex, 635) on the venous side of the oxygenator. Blood flows were 50 to 60 ml/kg/min (1000 to 1800 ml/min) for all’ perfusions. The circuit was primed with 700 to 1000 ml of a balanced salt solution (Travenol No. 11). Normothermia was maintained during the perfusions by (i) passing the tubing through a heated (42” C) water bath. (ii) securing a heating pad to the trunk of the animal, and (iii) directing an infrared heating lamp toward the area of the membrane oxygenator. Five-hour perfusions were the desired goal for all animals using a nonocclusive roller pump (Sarns). No blood transfusions were given, and no resuscitative efforts were made. Each animal received 600 ml of Ringer’s lactate during the 5-hr perfusions. Sampling times were baseline, then 1, 3, 5, and 6 hr. Systemic arterial pressure was monitored continuously for the 6 hr with Statham transducer (Model 267B) and recorded on an eight-channel Sanborn recorder (Model 350). Blood gases across the membrane oxygenator and from the animal were measured on a blood gas analyzer (Instrumentation Laboratories, Model 313). Platelet counts were determined with an electronic particle counter (Coulter, Model ZBI). The Quick one-stage method was used to determine the prothrombin time [ 181. The activated partial thrombinplastin time was performed with Celite-activated phospholipid (WarnerChilcott) by the method of Nye and Graham [15]. Whole blood clotting times were determined according to the technique of Lee and White [13]. Fibrinogen was measured by the turbidometic method of Parfentjev et al. [16]. Assessment of the fibrin split products was done by the staphyloccal clumping test of Hawiger and coworkers [6]. Serum hemoglobins were

VOL. 22, NO. 3, MARCH

1!?77

determined spectrophotometrically. Hematocrits were done by the microhematocrit technique. Oxygenator function was determined by comparing the prelung pressures and the postlung partial pressure of oxygen and blood flow during the perfusion to the baseline values for each parameter. There were two groups of animals. Group I (five animals) received systemic heparin (3 mg/kg initially and 1 mg/kg/hr during the perfusion). Protamine sulfate (one-half of the total heparin dose given) was used to reverse the heparin at the end of the perfusion. Group II (six animals) received no anticoagulants. Upon termination of the bypass (5 hr), the blood in the circuit was transfused into the animal. All blood remaining in the circuit was drained by gravity, and the circuit was immediately filled with a 10% neutral buffered formalin. Following a 3-day fixation period, all circuits were dismantled and examined for clots. Portions of the first, fifth, and tenth layers of the oxygenators were removed for examination by scanning electron microscopy. These materials were processed by dehydration through a graded series of ethanols and then ethanol-amyl acetate solutions and critical point dried in a Denton vacuum DCP-1 critical point dryer with liquid carbon dioxide. The sections then were attached to a copper stub by silver conducting paint and coated with a layer of 60-40 gold-palladium, approximately 25 mm thick. The specimens then were examined at 10 to 25 kV with a JEOL Model JSU-U3 scanning electron microscope. Photographs were taken with an accelerated voltage of 15 kv. Specific parameters looked for on these specimens were fibrin deposition, platelets, red blood cells, and white cells. RESULTS Blood Pressure

Systemic arterial pressures of the two groups are presented in Fig. 1. Systolic

FLETCHER,

MCKEE, AND HERMAN: MEMBRANE

OXYGENATION

275

200 180 r 160 ki 140 +I 120 5w 100 I -

80

;:;I;

3

0

5

6

HOURS FIG. 1. Systemic systolic arterial pressure during cardiopulmonary bypass represented as mm Hg (mean 2 SEM) for Groups I and II during the first 5 hr of perfusion and 1 hr following the completion of bypass. Group I received systemic heparinization and Group II had no anticoagulation.

pressures were higher in Group II throughout and following the perfusion; however, there were no significant differences between the groups. Coagulation State Coagulation parameters are presented in Table I. Thrombocytopenia is present throughout the perfusions in Group II; however, the lowest platelet count in Group II was only 75 x 103/mm3. There were significantly (P < 0.01) fewer platelets throughout the perfusion in Group II when compared to Group I. As expected, the most prolonged clotting times were seen in the systemic heparin group; however, all clotting times in Group II were significantly (P< 0.01) greater than the baseline value. Indeed, after 3 hr of perfusion the clotting times between Groups I and II were not statistically different. Prothrombin times and partial thromboplastin times were significantly (P < 0.005) greater than the baseline values in both groups. During perfusion, prothrombin times and partial thromboplastin times in Group II were significantly (P < 0.01) shorter than Group I values. The fibrin split products gradually increased with time in both groups during the perfusions, but were significantly (P < 0.01) higher in Group I when com-

pared to Group II values. Fibrinogen levels decreased with time in Groups I and II during the perfusions and were significantly less (P < 0.005) than the baseline values. There were no significant differences between the groups during perfusion, but 1 hr following the perfusion, Group II values were significantly (P < 0.01) less than Group I. Oxygenator Function Oxygenator function in Groups I and II is compared in Table II. The prelung pressure values in both groups were significantly (P < 0.05) less than the baseline values throughout the study, but there were no significant differences between the groups. Blood flows demonstrated some variation during the perfusions, but there were no significant differences between the groups. Postlung ~0, values minimally decreased with time in both groups; however, they were not significantly different from the baseline values. There were no significant differences between the groups. Hematocrit values in Groups I and II were significantly (P < 0.01) less than the baseline values at all sampling times during the perfusion; however, there were no significant differences between the groups. Serum hemoglobin values were significantly

JOURNAL OF SURGICAL RESEARCH: VOL. 22, NO. 3, MARCH 1977

276

TABLE 1 COAGULATION PARAMETERS (PERCENTAGE OF BASELINE [MEAN + SEMI) Time (hr) Baseline Parameter

Group

Platelets Clotting

time

F’rothrombin

time

Partial thromboplastin

time

3

1

5

6

103 k4 62 k 18

99 k 13 42 2 17”

98 k 12 34 2 6”

85 2 9 36 k 6”

4.7 2 0.2 (min)

>700 350 2 900

>700 483 e 160

>700 566 f 185

162 k 23 144 2 15

I II

12.9 + 0.15 (set)

681 k 20 130 f 1w

1181 + 160 139 f 14”

668 ? 73 146 + 16”

160 ? 8 188 2 24

I II

42 f 1.5 (se4

381 ” 32 123 k 7O

381 k 3 120 2 9”

396 k 6 130 k 11”

190 f 30 180 i 30

0.0 (lQ32)

467 f 33 170 2 7oa

566 k 33 233 ” 33”

600 ” 58 367 t 88a

350 t 100 500 + 160

70 * 1 62 2 6

69 k 8 56 2 8

68 f 8 50 t 6

89k 12 55 ? 5”

I II

275 + 33

I II

Fibrin split products

I II

Fibrinogen

I II

a Significantly

values

(x 103)

386 f 28 (mg/lOO ml)

less (P < 0.01).

(P < 0.001) greater at 1 hr in Group II (5.0 2 0.8 mg/lOO ml; mean ? SEM) when compared to Group I values (2.9 ? 0.62). Group I values were significantly (P < 0.01) greater at the 3-hr sampling time when compared to Group II. Interestingly, the mean serum hemoglobin did attain the highest level at the 5-hr sampling time in Group I (13.0 + 4 mg/lOO ml) but was not significantly different from the 5-hr Group II value (9.5 2 2.2 mg/lOO ml). There was no evidence of excessive bleeding from the incisions in either group during or following the perfusions. There were no gross thrombi present in any portion of the circuit from both groups of animals upon examination following bypass. Scanning Electron Microscopy Membrane Oxygenators

of the

The most striking finding was that there was no fibrin deposition on the surfaces of the oxygenators in animals perfused with systemic heparin (Group I). In two of the six animals in Group II, there was no fibrin deposition on any surface examined. In the other four animals the first layers

were free of fibrin, but the fifth and tenth layers showed minimal to moderate fibrin deposition. Both groups had platelets, red blood cells, and white blood cells present as well as an amorphous material covering the surfaces. The fifth and tenth layers of the oxygenators best represented the overall findings in each group and are presented in Figs. 2-5. These micrographs represent the worst-appearing fifth and tenth layers of the oxygenators in both groups. DISCUSSION

There are several interesting findings in this study. (i) Partial venovenous membrane oxygenation for 5 hr is possible in baboons without the use of any anticoagulant, and the results

are very

similar

to those

obtained

with dogs. The circuits utilized in these perfusions showed no gross evidence of thrombi and had no deterioration in membrane oxygenator performance. All the animals survived. (ii) The coagulation changes

during

perfusion

were

similar

in

animals that received heparin and those that did not. In comparison to the dog perfusions without

anticoagulants,

the

coagulation

FLETCHER,

MCKEE, AND HERMAN: MEMBRANE

OXYGENATION

277

TABLE 2 MEMBRANE OXYGENATOR FUNCTION (MEAN ? SEM)

Postlung blood gases

Baseline values

Prehmg pressures (% of baseline)

Blood flow (ml/kg/mm)

PH

PO, (mm Hg)

P’CQ (mm Hd

121 + 12 mm Hg

58 f 2

1.47 2 0.02

350 2 24

41 + 2

1 hr Group I (n = 5) Group II (n = 6)

87 k 9

53 2 2

7.45 2 0.02

320 it 59

42 k 2

90-r-7

58 2 3

7.54 f 0.02

291 2 43

34 t 2

3 hr Group I Group II

83 2 9 90*5

53 f 2 57 k I

7.43 2 0.04 7.51 ” 0.03

312 2 34 263 2 28

41 + 3 34 2 3

5 hr Group I Group II

80 2 8 80 2 5

53 2 2 60 ” 2

7.40 _’ 0.06 7.50 k 0.02

293 + 51 277 t 53

34 k 3 32 2 3

changes are qualitatively identical, however, in the baboon, the clotting times, PT, PTT, and platelet counts are less abnormal. (iii) When compared to the sur-

faces of the circuits used in nonanticoagulated dog perfusions, the circuits in the baboons appear to have more fibrin deposition. With systemic heparin anticoagula-

FIG. 2. Scanning electron microscope photograph of the fifth layer of the membrane oxygenator, Animal B-588, systemic heparinization (Group I). There are platelets and red blood cells present (original magnification, x 1500).

278

JOURNAL OF SURGICAL RESEARCH: VOL. 22, NO. 3, MARCH 1977

FIG. 3. Scanning electron microscope photograph, tenth layer of the membrane oxygenator, Animal B-11, systemic heparinization (Group I). There are platelets and red cells present with an occasional lymphocyte (original magnification, X 1500).

FIG. 4. Scanning electron microscope photograph, fifth layer of the membrane oxygenator, Animal B-62, no anticoagulation (Group II). Red blood cells are intertwined with fibrin strands. (original magnification, x 1500).

FLETCHER,

MCKEE, AND HERMAN: MEMBRANE

OXYGENATION

279

FIG. 5. Scanning electron microscope photograph, tenth layer of the membrane oxygenator, Animal B-23, no anticoagulation (Group II). Red blood cells and platelets are present in a loose fibrin meshwork (original magnification, x 1500).

tion, the circuits in the baboon experiments had much less debris on them than did the circuits used with systemic heparin in dog perfusions. There are several possible explanations for these findings. In baboons as in dogs there appears to be a process of “autoanticoagulation” that takes from 1 to 3 hr to develop. The extent of this autoanticoagulation in baboons is less than that in dogs. It is likely that consumption of clotting factors was occurring and continued throughout the perfusion until the stimulus (foreign surface) was removed at 5 hr. The absence of gross thrombi in the circuits at the end of the perfusion suggests that the stimulus to activate the clotting process was counterbalanced by the fibrinolytic system and/or the flow rates. There are no objective data in this study demonstrating that excessive clotting was present during the perfusions. In fact, normal oxygenator function implies that clotting was minimal. Although there was a coagu-

lopathy present in the nonanticoagulant group, the striking findings were that these changes were similar to those seen in fully heparinized baboons. The commonly held clinical view that “adequate” heparinization prevents a significant coagulopathy is not supported by these data. In fact, the coagulation parameters in the nonanticoagulated animals are better preserved when compared to the heparinized ones. The “anticoagulation” syndrome seen in the nonanticoagulated baboons is dangerous for clinical use. The addition of thoracotomy, cardiotomy suction, or associated disease states may well make the lesion fatal in man. The important finding in this study and the previous perfusion study in dogs is the similarity of the coagulation state between the fully heparinized and the nonanticoagulated group. These studies should stimulate us to look for better controlled and more specific agents to block the key components of the coagulation system.

280

JOURNAL OF SURGICAL RESEARCH: VOL. 22, NO. 3, MARCH 1977

The results of this study clearly demonstrate that (i) systemic heparin has no particular advantage over no heparin in parameters evaluated in this model, (ii) perfusions of baboons without anticoagulants can be done with results similar to those seen in dog perfusions; (iii) additional experiments in various species with and without anticoagulants must be done to determine the best guidelines for human cardiopulmonary bypass. ACKNOWLEDGMENTS We wish to thank Baxter-Travenol Laboratories for donating the membrane oxygenators used in this study. For technical assistance during the perfusions we want to thank Richard Battistelli and Frank Carmody. Others without whom this work could not have been completed were Kenneth Snyder, Bob Brown, David Villalas, Lorrita P. Watson, and Nancy Hage.

REFERENCES 1. Backman, F., et al. The hemostatic mechanism after open-heart surgery. J. Thorac. Cardiovnsc. Surg. 70: 76, 1975.

2. Bull, M. H., Huse, W. M., and Bull, B. S. Evaluation of tests used to monitor heparin therapy during estracorporeal circulation. Anesthesiology 43: 346, 1975. 3. Fletcher, J. R., McKee, A. E., Mills, M., Snyder, K. C., Herman, C. M. 24 Hour membrane oxygenation in dogs without anticoagulants. Surgery 80: 214, 1976. 4. Gibbon, J. H., Jr. Application of a mechanical heart and lung apparatus to cardiac surgery. Minn. Med. 37: 171, 1954. 5. Gralnick, H. R., and Fischer, R. D. The hemostatic response to open heart operations. J. Thorac. Cardiovasc. Surg. 61: 909, 1971. 6. Hawiger, J., et al. Measurement of fibrinogen and fibrin degradation products in serum by staphylococcal clumping test. J. Lab. C/in. Med. 75: 93, 1970. 7. Heiden, D., et al. Platelets, hemostasis, and thromboembolism during treatment of acute respiratory insufficiency with extracorporeal membrane oxygenation. J. Thorac. Cardiovasc. Surg. 70: 644, 1975. 8. Highsmith, R. F., and Rosenberg, R. D. The inhibition of human plasmin by human anti-

thrombin-heparin cofactor. J. Biol. Chem. 249: 4335, 1974. 9. Hill, J. D., et al. Acute respiratory insufficiency treatment with prolonged extracorporeal oxygenation. J. Thoruc. Curdiovasc. Surg. 64: 551, 1972. lo. Hill, J. D., et al. Clinical prolonged extracorporeal circulation for respiratory insufficiency: Hematologic effects. Trans. Amer. Sot. Artif. Intern. 18: 546, 1972. 1l, Organs Kolobow, T., et al. Extended term (to 16 days) partial extracorporeal blood gas exchange with the spiral membrane lung in unanesthetized lambs. Trans.

Amer.

Sot.

Artif.

Intern.

Organs

17:

350, 1971. 12 Lagergren, H., and Eriksson, J. C. Plastics with a stable surface monolayer of cross-linked heparin: Preparation and evaluation. Trans. Am. Sot. Artif. Intern. Organs 17: 10, 1971. ._ 13. Lee, R. D., and White P. D. A clinical study of the coagulation time of blood. Amer. J. Med. Sci. 145: 495, 1913. 14. Mattox, K. L., Guinn, G. A., Rubrio, P. A., et al. Use of activated coagulation time in intraoperative heparin reversal for cardiopulmonary operations. Ann. Thoruc. Surg. 19: 634, 1975. 15. Nye, S. W., and Graham, J. B. The partial thromboplastin time as a screening test for detection of latent bleeders, Amer. J. Med. Sci. 243: 279, 1962. 16. Parfentjev, I. A., Johnson, M. L., and Clifton, K. E. The determination of plasma fibrinogen by turbidity with ammonium sulfate. Arch. Biochem. Biophys. 46: 470, 1953. 17. Phillips, L. L., Malm, J. R., and Deterling, R. A. Coagulation defects following extracorporeal circulation. Ann. Surg. 157: 317, 1963. 18. Quick, A. J. Hemorrhagic Diseases, p. 991. Lea & Feabiger Philadelphia, 1966. 19. Ratliff, J. L., et al. Complications associated with membrane lung support by venoarterial perfusion. Ann. Ttiorac. Surg. 19: 537, 1975. 20. Roberts, W. E., and Morrow, A. G. Causes of early postoperative death following cardiac valve replacement. J. Thorac. Cardiovasc. Surg. 54: 422, 1%7. 21. Wakabayashi, A., et al. Clinical experience with heparinless venoarterial by pass without oxygenation for the treatment of acute cardiogenic shock. J. Thorac. Cardiovasc. Surg. 68: 687, 1974. 22. Wisch, N., et al. Hematologic complications of open heart surgery, Amer. J. Cardiol. 31: 282. 1973. 23. Wright, J. J., et al. Some advantages of the membrane oxygenator for open heart surgery. J. Thorac. Cardiovasc. Surg. 69: 884, 1975.