- Email: [email protected]
Total Lymphatic Irradiation and Bone Marrow in Human Heart Transplantation
Total Lymphatic Irradiation and Bone Marrow in Human Heart Transplantation Donald R. Kahn, M.D., Richard Hong, M.D., Alvin J. Greenberg, M.D., Enid F. Gilbert, M.D., Guillermo C. Dacumos, M.D., and John H. Dufek, P.A. ABSTRACT Six patients, aged 36 to 59 years, had heart transplants for terminal myocardial disease using total lymphatic irradiation (TLI) and donor bone marrow in addition to conventional therapy. All patients were poor candidates for transplantation because of marked pulmonary hypertension, unacceptable tissue matching, or age. Two patients are living and well more than four years after the transplants. Two patients died of infection at six and seven weeks with normal hearts. One patient, whose preoperative pulmonary hypertension was too great for an orthotopic heart transplant, died at 10 days after such a procedure. The other patient died of chronic rejection seven months postoperatively. Donor-specific tolerance developed in 2 patients. TLI and donor bone marrow can produce specific tolerance to donor antigens and allow easy control of rejection, but infection is still a major problem. We describe a new technique of administering TLI with early reduction of prednisone that may help this problem.
In 1953, Billingham and co-workers [11 induced tolerance to transplanted tissue in mice by injecting allogeneic bone marrow cells intravenously into newborn recipients. When allogeneic bone marrow cells are injected into adults, the cells are readily rejected unless the immune system of the adult has been eliminated by total body irradiation. Unless the match is perfect, graftversus-host disease develops and the new marrow destroys the host. Recently, Slavin and colleagues [2-51 achieved successful allogeneic bone marrow, skin, and heart transplantation without graft-versus-host disease in mice and rats after treating the recipients with fractionated total lymphatic irradiation (TLI).We [6] showed prolonged survival of heart and kidney allografts in unrelated mongrel dogs following TLI and donor bone marrow without other immunosuppression. However, achieving permanent allograft tolerance in large animals, such as dogs and monkeys, required additional immunosuppression. Six patients with terminal myocardial disease were chosen to receive TLI and donor bone marrow prior to From the Baptist Medical Center-Princeton, Birmingham, AL, and the University of Wisconsin Center for Health Sciences, Madison, WI. Accepted for publication Feb 10, 1984 Address reprint requests to Dr.Kahn, 817 Princeton Ave SW, Suite 300, Birmingham, AL 35211.
169
heart transplantation without marked alteration of conventional posttransplant therapy. These patients, although terminally ill with myocardial disease, were not candidates for a heart transplant according to accepted criteria because of age, pulmonary hypertension, or unacceptable tissue matching.
Methods Irradiation TLI is a well-known therapy for Hodgkin’s disease, and the methods of administering irradiation for immunosuppression and Hodgkin’s disease are similar. Using the mantle above the diaphragm and the inverted Y below the diaphragm, irradiation is targeted at the major lymph node groups (cervical, axillary, mediastinal, periaortic, and iliofemoral) as well as the thymus and spleen with lead shielding of nonlymphoid tissues. Three patients received 2,700 to 3,000 rads of TLI to the inverted Y followed by 2,700 to 3,000 rads to the mantle. Three patients received 2,700 rads to the inverted Y but only 180 to 1,400 rads to the mantle because of depressed white blood cell counts or clinical deterioration. One patient’s spleen was shielded during TLI; the spleen was later irradiated. TLI was given over a 39- to 49-day period followed by transplant 7 to 13 days later. Donor Bone Marrow At the time the donor heart was obtained, donor bone marrow was also aspirated from the iliac crest, placed in a container, and returned with the donor heart. After the transplants, each recipient was given 106 million to 11 billion cells of the donor bone marrow (average, 5 billion cells). The donor bone marrow was cleaned as little as possible so that all of the matter, including small spicules of marrow, was injected intraperitoneally. Technique of Heart Transplantation Two types of heart transplantation were performed. Four patients received heterotopic (double-heart) transplants because their pulmonary hypertension was too severe for the donor right ventricle to function against high pulmonary vascular resistance. The operative technique was similar to that described by Barnard and Losman [7], except that the inferior rather than the superior vena cava of the donor heart was anastomosed to the right atrium of the recipient heart. The donor heart was positioned between the recipient heart and the right lung. The donor and recipient hearts were connected in parallel with a side-to-side left atrial anastomosis, an
170 The Annals of Thoracic Surgery Vol38
No 2 August 1984
end-to-side aortic anastomosis, and an end-to-side pulmonary artery anastomosis. A donor aortic homograft was necessary to connect the donor and recipient pulmonary arteries. Two patients received typical orthotopic transplants, as described by Lower and Shumway 181.
Procurement of Donor Organs All donor hearts were procured from distant sites up to 1,000 miles away. The donor heart was removed and immediately irrigated with 1,000 ml of extracellular fluid [9] and stored in this solution at 4°C. Donor heart ischemic time ranged from two to five hours (i.e., the time from removal of the organ from the donor until the completion of the transplant when the aorta was unclamped and the heart was beating in its new recipient). The transplants were performed in Madison, WI; the most distant sites from which hearts were obtained were Trenton, NJ, and Dallas, TX. As experimental data have shown [9], hearts stored for five hours have the same function as hearts transplanted immediately. All hearts beat normally after one hour of recovery time. Other Conventional Immunosuppressant Therapy In addition to TLI, conventional immunosuppression was given. Azathioprine was initiated at a dose of 2 mg per kilogram of body weight per day and prednisone, 2 mglkg per day. Either Minnesota antilymphocyticglobulin or Stanford rabbit antithymocyte globulin was given to maintain the T lymphocyte count at less than 50 cells or E rosettes at less than 10% during the first three weeks. Maintenance of azathioprine depended on the white blood cell count. Because of leukopenia, the azathioprine was frequently stopped for a period and then resumed at a lower dose. Prednisone was reduced to 1 mglkg per day at three weeks and then 0.5 mglkg per day at three months except in 1 patient whose dosage was reduced to 0.3 mgkg per day at two weeks.
Results Two patients died owing to errors in judgment, so the evaluation of the effect of TLI on their courses is difficult. The first patient died of chronic rejection seven months after transplantation. She received 2,700 rads to the inverted Y but only 180 rads to the mantle because of increasing illness. She was doing well after her heterotopic transplant, but her condition suddenly deteriorated at seven months. This patient underwent our first heterotopic heart transplant and was not monitored by endomyocardial biopsies. The outcome demonstrates the necessity of performing frequent biopsies with the double-heart technique because of the difficulty in diagnosing rejection before it is too late. The second patient, a 47-year-old man, received an orthotopic transplant for terminal myocardial disease. He had tetralogy of Fallot and underwent a Potts anastomosis as a child and primary repair of the tetralogy 8 years prior to the transplant. His pulmonary vascular resistance was greater than anticipated. He had high right atrial pressures, nor-
mal left atrial pressures, and a borderline cardiac output from the beginning. A heterotopic rather than an orthotopic transplant should have been performed. He died 10 days postoperatively. Microscopic examination of his heart showed mild cellular infiltration of questionable importance. Two patients, 54 and 58 years old, received heterotopic transplants. They had uncomplicated courses until lethal infections developed, one at six weeks and the other at seven weeks. Neither of these patients had any rejection episodes. The white blood cells after TLI are extremely sensitive to azathioprine therapy. Prednisone was maintained at a dose of 2 mglkg per day for three to four weeks because it was necessary to markedly reduce the azathioprine dose. At the time of postmortem examination, both hearts were normal. Administration of high doses of prednisone after TLI appears to be especially lethal. Two patients are long-term survivors. The first is a 36year-old woman who received a heterotopic transplant in August, 1979, and is doing well 4% years later. She had previously had a mitral valve replacement for rheumatic disease and was seen by us with terminal myocardial disease, high pulmonary vascular resistance (she had a pulmonary artery pressure of 120 mm Hg before left ventricular deterioration), and marked tricuspid insufficiency with anasarca and ascites. In addition to a double-heart transplant, a tricuspid valve replacement was performed so that the patient’s own right ventricle could help support the right-sided circulation. She had positive T and B cell crossmatch. The T cell crossmatch was positive at the time of the transplant and at two weeks, but subsequently became negative at six and ten months. The B cell crossmatch was positive at the time of transplant and remained positive during the first year. The patient had one mild rejection episode two weeks after the transplant, which was quickly reversed by 15 mgkg per day of Minnesota antilymphocytic globulin given intravenously over 2 successive days. A specific tolerance to the donor developed, and the patient showed a much weaker mixed lymphocyte culture reaction to the donor than to a nonspecific third party (Table). She demonstrated a perfect physiological response from the heterotopic heart. The right ventricle of the heterotopic heart contributes 50% of the right-sided output, whereas the entire cardiac output of the left ventricle is from the donor heart. The donor heart has a
Specific Donor Tolerance in a Heterotopic Heart Transplant Recipient Days after Transplant
Culture
On Day of Transplant
52
73
134
Third-party MLC Donor MLC
4.6 x Not done
4.6~
4.6~
9 . 4 ~
< LOX
1 . 6 ~
2.2~
MLC
=
mixed lymphocyte culture.
171 Kahn et al: Total Lymphatic Irradiation and Bone Marrow in Human Heart Transplantation
normal ejection fraction on isotope studies. The patient is asymptomatic, working, and caring for her children. The second long-term survivor is a 44-year-old man who received an orthotopic transplant in May, 1980, and is doing well. The method of TLI was handled differently for two reasons. First, our [6] experimental data in mongrel dogs showed that the spleen was important in providing T suppressor cells and in achieving tolerance. Second, if azathioprine was maintained at adequate doses, prednisone dose levels could be reduced quickly to prevent major infections. This patient received 2,700 rads to the inverted Y, but with shielding of the spleen. He experienced hypersplenism with platelet counts of 20,000 per cubic millimeter. He then received 2,000 rads to the spleen over a 10-day period. After irradiation to the spleen, platelet count rose to 200,000/mm3.He was able to tolerate 2 mg/kg per day of azathioprine for the first three weeks with normal white blood cell and platelet counts. Prednisone was started at 2 mg/kg per day, reduced to 1 mg/kg per day by one week, and reduced further to 0.3 mg/kg per day by two weeks. This patient had several endomyocardial biopsies, all of which were negative. He has never had a rejection episode. In all of these patients, lymphocyte counts after TLI were reduced to 10 to 15% of normal and remained in this range for up to one year after transplantation. There was also a marked decrease in all the proliferative responses to phytohemagglutinin, concanavalin A, pokeweed mitogen, and alloantigens. The immunoglobulins seem to be relatively unaffected by TLI.
difficult group of patients. The 6 patients discussed in the present report also had complicated courses and could not be treated by any other therapy, including conventional heart transplantation. There are 2 longterm survivors, even though 1of the patients had a positive T and B cell crossmatch. Because of the reduced number of T lymphocytes after TLI, rejection episodes can be reversed easily with small doses of antithymocyte globulin. Infection is the main problem after TLI, particularly when large amounts of prednisone are used. Our experience with the last patient in this series may provide some answers.
References 1. Billingham RE, Brent L, Medawar PB: “Actively acquired tolerance” of foreign cells. Nature 172:603, 1953 2. Slavin S, Reitz B, Beiber CP, et al: Transplantation tolerance
3. 4.
5. 6.
Comment Some of the first human renal transplant trials of TLI were done at the University of Minnesota in uremic patients who had previously rejected one or more kidney grafts [101. With conventional immunosuppression in patients having retransplantation, one- and four-year survival was 46 and 28%, respectively. In 22 patients treated with TLI, the one- and two-year graft survival was 79 and 74%; these figures demonstrate considerable improvement over conventional therapy alone in this
7. 8. 9.
10.
in adult rats using total lymphoid irradiation: permanent survival of skin, heart, and marrow allografts. J Exp Med 147:700, 1978 Slavin S, Strober S, Fuks Z , Kaplan HS: Use of total lymphoid irradiation in tissue transplantation in mice. Transplant Proc 9:1001, 1977 Slavin S, Strober S, Fuks Z, Kaplan HS: Induction of specific tissue transplantation tolerance using fractionated total lymphoid irradiation in adult mice: long-term survival of allogeneic bone marrow and skin grafts. J Exp Med 14634, 1977 Slavin S, Fuks Z, Kaplan HS, Strober S: Transplantation of allogeneic bone marrow without graft-versus-host disease using total lymphoid irradiation. J Exp Med 147963, 1978 Kahn DR, Dufek JH, Hong R, et al: Heart and kidney transplantation using total lymphoid irradiation and donor bone marrow in mongrel dogs. J Thorac Cardiovasc Surg 80:125, 1980 Bamard CN, Losman JG: Left ventricular bypass. S Afr Med J 49:303, 1975 Lower RR, Shumway NE: Studies on orthotopic homotransplantation of the canine heart. Surg Forum 11:18, 1960 Swanson DK, Dufek JH, Kahn DR Myocardial preservation for transplantation. Transplant Proc II:1478, 1979 Najarian JS, Sutherland DER, Ferguson RM, et al: Total lymphoid irradiation and kidney transplantation: a clinical experience. Transplant Proc 13:417, 1981
In 1953, Billingham and co-workers [11 induced tolerance to transplanted tissue in mice by injecting allogeneic bone marrow cells intravenously into newborn recipients. When allogeneic bone marrow cells are injected into adults, the cells are readily rejected unless the immune system of the adult has been eliminated by total body irradiation. Unless the match is perfect, graftversus-host disease develops and the new marrow destroys the host. Recently, Slavin and colleagues [2-51 achieved successful allogeneic bone marrow, skin, and heart transplantation without graft-versus-host disease in mice and rats after treating the recipients with fractionated total lymphatic irradiation (TLI).We [6] showed prolonged survival of heart and kidney allografts in unrelated mongrel dogs following TLI and donor bone marrow without other immunosuppression. However, achieving permanent allograft tolerance in large animals, such as dogs and monkeys, required additional immunosuppression. Six patients with terminal myocardial disease were chosen to receive TLI and donor bone marrow prior to From the Baptist Medical Center-Princeton, Birmingham, AL, and the University of Wisconsin Center for Health Sciences, Madison, WI. Accepted for publication Feb 10, 1984 Address reprint requests to Dr.Kahn, 817 Princeton Ave SW, Suite 300, Birmingham, AL 35211.
169
heart transplantation without marked alteration of conventional posttransplant therapy. These patients, although terminally ill with myocardial disease, were not candidates for a heart transplant according to accepted criteria because of age, pulmonary hypertension, or unacceptable tissue matching.
Methods Irradiation TLI is a well-known therapy for Hodgkin’s disease, and the methods of administering irradiation for immunosuppression and Hodgkin’s disease are similar. Using the mantle above the diaphragm and the inverted Y below the diaphragm, irradiation is targeted at the major lymph node groups (cervical, axillary, mediastinal, periaortic, and iliofemoral) as well as the thymus and spleen with lead shielding of nonlymphoid tissues. Three patients received 2,700 to 3,000 rads of TLI to the inverted Y followed by 2,700 to 3,000 rads to the mantle. Three patients received 2,700 rads to the inverted Y but only 180 to 1,400 rads to the mantle because of depressed white blood cell counts or clinical deterioration. One patient’s spleen was shielded during TLI; the spleen was later irradiated. TLI was given over a 39- to 49-day period followed by transplant 7 to 13 days later. Donor Bone Marrow At the time the donor heart was obtained, donor bone marrow was also aspirated from the iliac crest, placed in a container, and returned with the donor heart. After the transplants, each recipient was given 106 million to 11 billion cells of the donor bone marrow (average, 5 billion cells). The donor bone marrow was cleaned as little as possible so that all of the matter, including small spicules of marrow, was injected intraperitoneally. Technique of Heart Transplantation Two types of heart transplantation were performed. Four patients received heterotopic (double-heart) transplants because their pulmonary hypertension was too severe for the donor right ventricle to function against high pulmonary vascular resistance. The operative technique was similar to that described by Barnard and Losman [7], except that the inferior rather than the superior vena cava of the donor heart was anastomosed to the right atrium of the recipient heart. The donor heart was positioned between the recipient heart and the right lung. The donor and recipient hearts were connected in parallel with a side-to-side left atrial anastomosis, an
170 The Annals of Thoracic Surgery Vol38
No 2 August 1984
end-to-side aortic anastomosis, and an end-to-side pulmonary artery anastomosis. A donor aortic homograft was necessary to connect the donor and recipient pulmonary arteries. Two patients received typical orthotopic transplants, as described by Lower and Shumway 181.
Procurement of Donor Organs All donor hearts were procured from distant sites up to 1,000 miles away. The donor heart was removed and immediately irrigated with 1,000 ml of extracellular fluid [9] and stored in this solution at 4°C. Donor heart ischemic time ranged from two to five hours (i.e., the time from removal of the organ from the donor until the completion of the transplant when the aorta was unclamped and the heart was beating in its new recipient). The transplants were performed in Madison, WI; the most distant sites from which hearts were obtained were Trenton, NJ, and Dallas, TX. As experimental data have shown [9], hearts stored for five hours have the same function as hearts transplanted immediately. All hearts beat normally after one hour of recovery time. Other Conventional Immunosuppressant Therapy In addition to TLI, conventional immunosuppression was given. Azathioprine was initiated at a dose of 2 mg per kilogram of body weight per day and prednisone, 2 mglkg per day. Either Minnesota antilymphocyticglobulin or Stanford rabbit antithymocyte globulin was given to maintain the T lymphocyte count at less than 50 cells or E rosettes at less than 10% during the first three weeks. Maintenance of azathioprine depended on the white blood cell count. Because of leukopenia, the azathioprine was frequently stopped for a period and then resumed at a lower dose. Prednisone was reduced to 1 mglkg per day at three weeks and then 0.5 mglkg per day at three months except in 1 patient whose dosage was reduced to 0.3 mgkg per day at two weeks.
Results Two patients died owing to errors in judgment, so the evaluation of the effect of TLI on their courses is difficult. The first patient died of chronic rejection seven months after transplantation. She received 2,700 rads to the inverted Y but only 180 rads to the mantle because of increasing illness. She was doing well after her heterotopic transplant, but her condition suddenly deteriorated at seven months. This patient underwent our first heterotopic heart transplant and was not monitored by endomyocardial biopsies. The outcome demonstrates the necessity of performing frequent biopsies with the double-heart technique because of the difficulty in diagnosing rejection before it is too late. The second patient, a 47-year-old man, received an orthotopic transplant for terminal myocardial disease. He had tetralogy of Fallot and underwent a Potts anastomosis as a child and primary repair of the tetralogy 8 years prior to the transplant. His pulmonary vascular resistance was greater than anticipated. He had high right atrial pressures, nor-
mal left atrial pressures, and a borderline cardiac output from the beginning. A heterotopic rather than an orthotopic transplant should have been performed. He died 10 days postoperatively. Microscopic examination of his heart showed mild cellular infiltration of questionable importance. Two patients, 54 and 58 years old, received heterotopic transplants. They had uncomplicated courses until lethal infections developed, one at six weeks and the other at seven weeks. Neither of these patients had any rejection episodes. The white blood cells after TLI are extremely sensitive to azathioprine therapy. Prednisone was maintained at a dose of 2 mglkg per day for three to four weeks because it was necessary to markedly reduce the azathioprine dose. At the time of postmortem examination, both hearts were normal. Administration of high doses of prednisone after TLI appears to be especially lethal. Two patients are long-term survivors. The first is a 36year-old woman who received a heterotopic transplant in August, 1979, and is doing well 4% years later. She had previously had a mitral valve replacement for rheumatic disease and was seen by us with terminal myocardial disease, high pulmonary vascular resistance (she had a pulmonary artery pressure of 120 mm Hg before left ventricular deterioration), and marked tricuspid insufficiency with anasarca and ascites. In addition to a double-heart transplant, a tricuspid valve replacement was performed so that the patient’s own right ventricle could help support the right-sided circulation. She had positive T and B cell crossmatch. The T cell crossmatch was positive at the time of the transplant and at two weeks, but subsequently became negative at six and ten months. The B cell crossmatch was positive at the time of transplant and remained positive during the first year. The patient had one mild rejection episode two weeks after the transplant, which was quickly reversed by 15 mgkg per day of Minnesota antilymphocytic globulin given intravenously over 2 successive days. A specific tolerance to the donor developed, and the patient showed a much weaker mixed lymphocyte culture reaction to the donor than to a nonspecific third party (Table). She demonstrated a perfect physiological response from the heterotopic heart. The right ventricle of the heterotopic heart contributes 50% of the right-sided output, whereas the entire cardiac output of the left ventricle is from the donor heart. The donor heart has a
Specific Donor Tolerance in a Heterotopic Heart Transplant Recipient Days after Transplant
Culture
On Day of Transplant
52
73
134
Third-party MLC Donor MLC
4.6 x Not done
4.6~
4.6~
9 . 4 ~
< LOX
1 . 6 ~
2.2~
MLC
=
mixed lymphocyte culture.
171 Kahn et al: Total Lymphatic Irradiation and Bone Marrow in Human Heart Transplantation
normal ejection fraction on isotope studies. The patient is asymptomatic, working, and caring for her children. The second long-term survivor is a 44-year-old man who received an orthotopic transplant in May, 1980, and is doing well. The method of TLI was handled differently for two reasons. First, our [6] experimental data in mongrel dogs showed that the spleen was important in providing T suppressor cells and in achieving tolerance. Second, if azathioprine was maintained at adequate doses, prednisone dose levels could be reduced quickly to prevent major infections. This patient received 2,700 rads to the inverted Y, but with shielding of the spleen. He experienced hypersplenism with platelet counts of 20,000 per cubic millimeter. He then received 2,000 rads to the spleen over a 10-day period. After irradiation to the spleen, platelet count rose to 200,000/mm3.He was able to tolerate 2 mg/kg per day of azathioprine for the first three weeks with normal white blood cell and platelet counts. Prednisone was started at 2 mg/kg per day, reduced to 1 mg/kg per day by one week, and reduced further to 0.3 mg/kg per day by two weeks. This patient had several endomyocardial biopsies, all of which were negative. He has never had a rejection episode. In all of these patients, lymphocyte counts after TLI were reduced to 10 to 15% of normal and remained in this range for up to one year after transplantation. There was also a marked decrease in all the proliferative responses to phytohemagglutinin, concanavalin A, pokeweed mitogen, and alloantigens. The immunoglobulins seem to be relatively unaffected by TLI.
difficult group of patients. The 6 patients discussed in the present report also had complicated courses and could not be treated by any other therapy, including conventional heart transplantation. There are 2 longterm survivors, even though 1of the patients had a positive T and B cell crossmatch. Because of the reduced number of T lymphocytes after TLI, rejection episodes can be reversed easily with small doses of antithymocyte globulin. Infection is the main problem after TLI, particularly when large amounts of prednisone are used. Our experience with the last patient in this series may provide some answers.
References 1. Billingham RE, Brent L, Medawar PB: “Actively acquired tolerance” of foreign cells. Nature 172:603, 1953 2. Slavin S, Reitz B, Beiber CP, et al: Transplantation tolerance
3. 4.
5. 6.
Comment Some of the first human renal transplant trials of TLI were done at the University of Minnesota in uremic patients who had previously rejected one or more kidney grafts [101. With conventional immunosuppression in patients having retransplantation, one- and four-year survival was 46 and 28%, respectively. In 22 patients treated with TLI, the one- and two-year graft survival was 79 and 74%; these figures demonstrate considerable improvement over conventional therapy alone in this
7. 8. 9.
10.
in adult rats using total lymphoid irradiation: permanent survival of skin, heart, and marrow allografts. J Exp Med 147:700, 1978 Slavin S, Strober S, Fuks Z , Kaplan HS: Use of total lymphoid irradiation in tissue transplantation in mice. Transplant Proc 9:1001, 1977 Slavin S, Strober S, Fuks Z, Kaplan HS: Induction of specific tissue transplantation tolerance using fractionated total lymphoid irradiation in adult mice: long-term survival of allogeneic bone marrow and skin grafts. J Exp Med 14634, 1977 Slavin S, Fuks Z, Kaplan HS, Strober S: Transplantation of allogeneic bone marrow without graft-versus-host disease using total lymphoid irradiation. J Exp Med 147963, 1978 Kahn DR, Dufek JH, Hong R, et al: Heart and kidney transplantation using total lymphoid irradiation and donor bone marrow in mongrel dogs. J Thorac Cardiovasc Surg 80:125, 1980 Bamard CN, Losman JG: Left ventricular bypass. S Afr Med J 49:303, 1975 Lower RR, Shumway NE: Studies on orthotopic homotransplantation of the canine heart. Surg Forum 11:18, 1960 Swanson DK, Dufek JH, Kahn DR Myocardial preservation for transplantation. Transplant Proc II:1478, 1979 Najarian JS, Sutherland DER, Ferguson RM, et al: Total lymphoid irradiation and kidney transplantation: a clinical experience. Transplant Proc 13:417, 1981