Bell, PRF

Brettschneider, L

Martin, AJ

Starzl, TE

Tarr, JS

Taylor, PD

English

PubMed

Name not available

Conservation of the liver

Bell, PR

D-Scholarship@Pitt

Conservation of the Liver By LAWRENCE BRETTSCHNEIDER, PETER R. F. BELL, ALFRED J. MARTIN, JR., JOHN S. TARR, PAUL D. TAYLOR AND THOMAS E. STARZL DURING the past two years, a system of isolated ex-vivo liver preservation has been developed which combines the use of hy-pothermia, hyperbaric oxygenation, and low flow perfusion with fresh diluted blood.1 The effectiveness was measured by the ability of the organs to sustain life both acutely and chronically when transplanted as orthotopic homografts in immuno-suppressed canine recipients. Uniform suc-cess was obtained following storage for 8 to mf hours, and in 60% of the experiments for as long as 25 hours. The same technique has been successfuly employed in the pres-ervation of 11 human livers for up to T~ hours prior to orthotopic transplantation, but the need for fresh homologous blood in this system is inconvenient in the clinical setting. Consequently a study was under-taken to evaluate various acellular perfus-ates which could be made readily available, including an oncotically balanced salt solu-tion, a hemoglobin solution, unaltered plasma, and the desilted plasma described by Belzer.2 METHODS The basic experimental model was the same as previously described.1 In summary, mongrel donor dogs were cooled to 32° to 34° C and sacrificed at the time of hepatectomy. The donor organ was cleared of blood, and further cooled by an intra-vascular infusion with a chilled (4°C) balanced salt solution. The organ was then placed in a refrigerated (4° C) hyperbaric oxygen chamber which was pressurized to 40 PSIG over 20 minutes. From the Department of Surgery, University of Colorado School of Medicine and Veterans Admin-istration Hospital, Denver, Colorado. Supported by United States Public Health Ser-vice grants AM-06344, HE-07735, AM-07772, AI-04152, FR-00051, FR-00069, F05-TW-1l54, AM-12148 and AI-AM-08898. The portal vein and hepatic artery were continu-ously perfused with the appropriate solution at a flow rate of 4.8 and 1.2 milliliter/gram liver/hour, respectively, and stored for 8 to 25 hours. The chamber was then decompressed over 3 to 4 hours. Orthotopic transplantation was performed to non-related mongrel dogs who received an immuno-suppression regimen consisting of heterologous antilymphocyte globulin (ALG), azathioprine, and prednisone. Only the perfusate was varied in the different experimental groups as indicated below. Group I-Fresh homologous blood was added to an equal volume of a buffered (pH 7.4-7.5) balanced salt solution containing 5.0 gm % low molecular weight dextran, 150 mgm % glucose, 2 milliequivalents per liter magnesium sulfate, 50 mgm per liter procaine and 100 mgm per liter heparin. Group 2-The balanced salt solution containing low molecular weight dextran solution described in Group 1 was employed without the addition of blood. Group 3-Fresh hemoglobin solution was pre-pared by Folkman's modification (3) of Pennell's technique ( 4) . One part of the balanced and oncotically controlled buffered salt solution de-scribed above was added to 3 parts hemoglobin solution, resulting in a final hemoglobin concentra-tion of 5 to 7 gm %. The pH and electrolyte compo-sition was adjusted to that of normal whole blood. Group 4-Pooled fresh heparinized homologous plasma was added to an equal volume of the di-luting solution described in Group 1. Group 5-Pooled fresh heparinized plasma was frozen for 12 hours, quickly thawed and passed through a series of millipore filters to remove the lipoprotein flocculate as described by Belzer.2 This perfusate was not diluted. RESULTS Group 1-As previously reported,15livers perfused for U-9~ hours with diluted fresh homologous blood had an average weight loss of -4.2%, (range +4.6 to -12.5%), and inevitably functioned well immediately following transplantation.1 All 5 recipients of these organs survived the operation and 132 TRANSPLANTATION PROCEEDINGS, VOL. I, No.1 (MARCH), 1969. 133 Table 1.-Experimental Groups AVERAGE OPERATIVE SURVIVAL NUMBER AVASCULAR POST-PERFUSION SURVIVAL GREATER OF PERIOD WEIGHT GAIN (24 hours) THAN GROUP PERFUSATE EXPERIMENTS (Hours) (%) 5 DAYS Controls ----------- 12 ~-1~ ----------- 12/12 11/12 lA Diluted Blood 5 U-9~ -4.2 5/ 5 5/ 5 IB Diluted Blood 5 O4~-OR% 4.3 3/ 5 3/ 5 2 Asanguinous 5 O1~ - O4~ -1.9 1/5 1/ 5 3 Diluted hemo- 5 globin solution 4 Diluted plasma 5 5 Desilted plasma 5 lived for more than 5 days (Table 1, Group lA). Chronic survival did not significantly differ from a series of control experiments in which transplantation was carried out after core cooling without a period of in-terim preservation (Table 1, controls). When the preservation period was ex-tended to 24-25 hours, the 5 organs had an average weight gain of 4.3% (range + 1.9 to 9.6% ). Two of the 5 recipients died within 24 hours from an uncontrollable hemor-rhagic diathesis. The remaining 3 animals lived for 8, 46 and 128 days (Table 1, GrouplB). The pH of the perfusate remained within normal limits during the U-9g~ hour experi-ments and minimal addition of buffering agents was required in the 24 hour experi-ments (Table 2). Group 2-Five homografts perfused with an asanguinous solution for between 21 to 24 hours had an average weight loss of -1.9% (range - 9.1 to + 9.4% ). Moder-ately severe acidosis developed during stor-age with occasional intraperfusion reduc-tions in the pH to as low as 7.10 in spite of active attempts at buffering (Table 2). Four of the 5 animals receiving these or-gans died of hepatic insufficiency and hem-orrhage within 24 hours. The fifth lived for 9 days with persistently poor liver function. Group 8-The 5 livers perfused with diluted hemoglobin solution for 8 to 1M~ hours had an average weight loss of -0.7% (range +5.4 to -7.9%). During preser-U-1M~ -0.7 1/ 5 0/ 5 9~ -Ire; --0.9 3/ 5 2/ 5 9~-11 -9.4 0/ 5 0/ 5 vation, the perfusate invariably became al-kalotic (Table 2). Of the 5 dogs receiving these organs, 4 died of hemordlage and hepatic insufficiency on the day of opera-tion. The remaining recipient demonstrated poor hepatic function and died of intus-sception on the 5th postoperative day (Ta-ble 1). Group 4-Five livers perfused with di-luted unaltered plasma had an average weight loss of ---0.9% (range + 12.2 to - 6.6%) following preservation for 9~ to 1~ hours. Acid-base relationships remained normal during perfusion (Table 2). Two re-cipients died within 24 hours of orthotopic transplantation, one from hemorrhage and the other with extensive ascites and atelec-tasis. Three animals survived operation, one dying after two days from hemorrhage and hepatic insufficiency. The remaining two recipients survived for 19 days with death due to homograft rejection in one case, and a perforated duodenal ulcer in the presence of good liver function in the other. Group 5-Desilted plasma was used to perfuse 5 livers for 9* to 11 hours with an average post-preservation weight loss of -9.4% (range from -5.4 to -16.3%). Prior to preservation, the osmolarity of their perfusate was low but increased during per-fusion at a rate much greater than seen in the other experiments (Table 2). In addi-tion, the specially prepared plasma tended to be acidotic prior to perfusion with further Table 2.-Analysis of Pre-Preservation and Post-Preservation Perfusates Plasma (mgm NA (meq/L) K (meq/L) Cl (meq/L) Osmolarity (mOsm) pH Hemoglobin % ) GROUPS PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST 1 A 142±1.8 139± 2.2 4.8±1.2 11.2±3.1 133± 6.3 135± 3.1 321± 5.8 326± 2.5 7.46± .09 7.35± .01 1.8±2.1 2.3±0.92 1 B 146±1.8 146± 1.8 4.8±.42 9.4±5.7 130± 6.8 133± 3.6 319± 5.6 330±13.6 7.48± .03 "'7.38± .03 1.8±1.0 10.0±9.8 2 145±1.9 150± 4.9 5.3±0.8 1O.8±1.3 135± 1.3 138± 3.0 318± 8.5 339±11.7 7.41±0.4 3 134±6.9 139±14.8 4.5± .3 11.9±3.1 104±20.2 107±15.3 272±20.7 288±16.7 7.45±0.1 4 149±2.4 147± 1.4 3.4±0.5 5.3±2.9 111± 4.1 122± 8.5 311± 6.7 335± 9.2 7.43±0.05 5 138±1.6 138± 2.9 4.4±9.4 1O.1±.65 69± 1.9 78± 2.9 263± 1.6 336± 5.3 7.31± .35 '" Average intrapreservation buffering per experiment: Group 1B: Tromethamine 144 mgm, NaHCOa 1.78 meq Group 2 : Tromethamine 648 mgm, NaCHO, 7.12 meq Mean values and standard deviation "'7.29±1.3 7.64±0.7 7.40±0.07 7.21±0.01 I-' ~ reductions in pH developing during storage. All 5 recipients died within 24 hours follow-ing transplantation, of hemorrhage, ascites and/ or acute hepatic insufficiency. DISCUSSION Throughout the developmental period of the preservation technique used in these experiments, a systematic evaluation has shown that elimination of hyperbaria, rapid decompression of the hyperbaric chamber, omission of perfusion or reduction in the rate of blood flow yielded inferior results when the preserved organs were tested as orthotopic homografts.1 In addition, the ben-eficial effect of hypothermia in reducing the ischemic injury to a hepatic graft prior to revascularization is well documented.5 ,6 When fresh homologous blood was used as the perfusing solution, well preserved livers were uniformly obtained following storage for 8 to 9g~ hours and frequently for as long as 25 hours. However, the use of fresh blood has its disadvantages. The po-tential hazard of hemolysis and resulting tissue damage is always present and the necessity for compatible blood in the clini-cal application of this technique may pre-clude its use if a suitable blood donor is not readily available. Consequently, various acellular solutions were tested as the only variable in the storage system. Perfusion with oncotically controlled buffered balanced salt solution, hemoglobin solution, unaltered plasma, and desilted plasma produced livers of poor quality which were generally unable to sustain life following transplantation (Table 1). The explanation for the inferior results obtained with acellular perfusates is not clear. Upon revascularization, homogeneous perfusion of the graft was invariably obtained and the development of outflow block was almost never encountered. Nevertheless, most of the recipients developed a hemorrhagic di-athesis within 30 minutes following re-establishment of blood flow and/ or exhib-135 ited severe hepatic insufficiency manifest by the development of massive ascites, "third-space" sequestration, and failure to recover from anesthesia. A frequently proposed explanation for the graft injury caused by prolonged organ perfusion has been the development of pro-gressively rising intravascular resistance and edema with consequent tissue dam-age.2 ,7,S This explanation does not appear to be applicable to our system, as we have shown that organ edema, at least as mea-sured by weight gain, can be virtually elim-inated with hyperbaric oxygenation and slow terminal decompression. In the experi-ments herein described, significant weight gain was rarely seen regardless of the per-fusate used (Table 1). The importance of maintaining a normal acid-base relationship during storage of iso-lated organs has been frequently empha-sized.7 ,9 Buckberg et al,lO Eiseman et al ll and others 12,13.14 have demonstrated the accumulation of acid metabolites in the ef-fluent of livers subjected to minimal anoxia and/ or isolated perfusion. The development of progressive acidosis during preservation with the asanguinous perfusate reflects the inefficient buffering capacity of non-blood containing solutions in a recirculating sys-tem and may explain the poor tesults ob-tained in Group 2. Hemoglobin was added to the asanguin-ous perfusate (Group 3) in an effort to provide improved buffering capacity and better oxygenation during the initial cool-ing phase in the chamber. A high perfusate p02 was invariably obtained. Instead of de-veloping acidosis as with the other acellular solutions, the perfusate now tended to be-come progressively alkalotic. The explana-tion for this finding is not clear, nor is it evident why there was uniform graft failure in this group of experiments. The results obtained with unaltered ho-mologous plasma (Group 4) were better than with the other acellular perfusates but 136 mediocre when compared to the blood per-fusions. Therefore, a final group of experi-ments (Group 5) were performed using lipoprotein depleted plasma, shown by Bel-zer to improve the results in his plasma per-fusion system for kidney preservation. This solution differed from the other perfusates in two important ways, the pH and osmo-larity being significantly lower (Table 2). During perfusion with Belzer's solution the pH tended to decrease further, resulting in a degree of acidosis significantly greater than that seen with the other perfusates. Although the final pH was lower, the de-gree of change during perfusion was the same, suggesting that the buffering capacity of the desilted plasma was not inferior to the other perfusate containing unaltered plasma. The osmolarity markedly increased during the interval of storage, a fact which is not surprising in view of the prior re-moval of lipoproteins which would predict-ably lower the oncotic pressure of the solution. It has been suggested that oncotic pressure is not necessarily important in renal preservation.7 However, the large electro-lyte shifts occuring with the desilted plasma may have a deleterious effect dur-ing liver storage, accounting in part for the almost invariable graft failure seen in this group. In view of the poor results obtained with the acellular solutions tested in our preser-vation system, it would seem logical to con-tinue using fresh homologous blood as the perfusate. Although occasional recipient sur-vival was obtained with unaltered plasma, there was almost always evidence of moder-ate to marked hepatic insufficiency manifest by a bleeding diathesis following ortho-topic transplantation. However, in the ap-plication of preservation to human liver transplantation, only a system providing repeated survival can be justified. As fresh homologous blood is the only perfusate pro-viding a high degree of reliability in our system, its continued use would appear to be advisable. SUMMARY A system of whole organ liver preservation has been developed and tested by means of transplanting the organs as orthotopic homografts. The separate components of hypothermia, hyperbaric oxygenation and continuous low How blood perfusion have been shown to contribute to the efficiency of the method in which uniform success was obtained following storage for 8 to g~ hours and often for as long as 25 hours. Various acellular perfusates were tested as the only variable in the storage technique. Perfusion with oncotically controlled buffered bal-anced salt solution, hemoglobin solution, unaltered plasma or lipiprotein depleted plasma yielded inferior results when com-pared with the original diluted blood per-fusate. REFERENCES 1. Brettschneider, L., Daloze, P. m., Huguet, C., Porter, K. A., Groth, C. G., Kashiwagi, N., Hutchi-son, D. E. and Starzl, T. E.: The use of combined preservation techniques for extended storage of orthotopic liver homografts. Surg. Gyn. Obst. 126: 263,1968. 2. Belzer, F. 0., Ashby, B. S., Huang, J. S. and Dunphy, J. E.: Etiology of rising perfusion pres-sure in isolated organ perfusion. Ann. Surg. 168: 382, 1968. 3. Follanan, J., Long, D. M., Jr. and Becker, F. F.: Growth and metastasis of tumor in organ cul-ture. Cancer 16:453, 1963. 4. Pennell, R B. and Smith, W. E.: Preparation of stabilized solutions of hemoglobin. Blood 4:380, 1949. 5. Moore, F. D., Wheele, H. B., Demissianos, H. V., Smith, L. L., Balantura, 0., Abel, K., Green-berg, J. B. and Dammin, G. J.: Experimental whole organ transplantation of the liver and of the spleen. Ann. Surg. 152:374, 1960. 6. Starzl, T. E., Kaupp, H. A., Brock, D. R, Lazarus, R E. and Johnson, R U.: Reconstruc-tive problems in canine liver homotransplantation with special reference to the postoperative role of hepatic venous How. Surg. Gyn. Obst. 111:733, 1960. 7. Humphries, A. L., Jr.: Organ Preservation: A review. Transplantation 5:1138,1967. 8. Huntley, R., Homatas, J. and Eiseman, B.: The role of hyperbarism in isolated organ per-fusion. In Organ Perfusion and Preservation. Ap-pleton, Century, and Grofts, New York, 1968. (In press ). 9. Ackermann, J. R. W. and Barnard, C. W.: Successful storage of kidneys. Brit. J. Surg. 53:525, 1966. 10. Buckberg, G. D., Ono, H., Joseph, W. L., Tocornal, J. A., Fonkalsrud, E. W. and Longmire, W. P., Jr.: Hypotension following revascularization of the anoxic liver: Factors influencing its occur-rence and prevention. Surgery 63:446, 1968. 137 11. Eiseman, B., Knipe, P., Koh, Y., Normell, L., and Spencer, F. c.: Factors affecting hepatic vas-cular resistance in the perfused liver. Ann. Surg. 157:532, 1963. 12. Barnett, W.O., Turner, M. D., and Walker, J. W.: The effect of afferent circulatory arrest upon hydrogen ion concentration in the liver. Surg. Gyn. Obst. 106:511, 1958. 13. Jolly, P. C., and Foster, J. H.: Hepatic in-flow stasis. Surgery 54:45, 1963. 14. Kestens, P. J., Farrelly, J. A. and McDer-mott, W. V., Jr.: A technique of isolation and per-fusion of the canine liver. J. S. Res. 1:58, 1961. 

Conservation of the liver.

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Conservation of the liver.

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