1 European Journal of Cardio-thoracic Surgery 31 (2007) Blood type incompatible cardiac transplantation in young infants Sabine H. Daebritz a, *,1, Michael Schmoeckel a,1, Helmut Mair a, Rainer Kozlik-Feldmann b, Georg Wittmann c, Christian Kowalski d, Ingo Kaczmarek a, Bruno Reichart a a Department of Cardiac Surgery, Klinikum Grosshadern, Ludwig-Maximilians-University, Munich, Germany b Department of Pediatric Cardiology, Klinikum Grosshadern, Ludwig-Maximilians-University, Munich, Germany c Department of Transfusion Medicine, Klinikum Grosshadern, Ludwig-Maximilians-University, Munich, Germany d Department of Anesthesiology, Klinikum Grosshadern, Ludwig-Maximilians-University, Munich, Germany Received 14 June 2006; received in revised form 22 November 2006; accepted 24 November 2006; Available online 17 January 2007 Abstract Objective: Donor organ shortage in pediatric heart transplantation (HTx) is causing mortality rates of 30 50% on the waiting list. Due to immaturity of the immune system of newborns and infants, ABO-incompatible HTx may be an option to increase donor availability. We present our experience with ABO-incompatible HTx. Methods: Three infants were transplanted ABO-incompatible since 12/2004: (1) hypoplastic left heart complex, (2) restrictive hypertrophic cardiomyopathy, (3) dilative cardiomyopathy. Age at HTx was 7, 5, and 3.5 months. All recipients had blood type O, donors were A, A, and B. Informed consent was given by parents, the ethics committee, and Eurotransplant. Results: Preoperative isohemagglutinin titers were low (Patient 1: 1:4 for anti-a1, A2, B, Patient 2: 1:4, 1:1, 1:4 for anti-a1, A2, B, respectively, and Patient 3: 0 for all, but quick spin 1+ for all). Intraoperatively, plasma was separated from red blood cells and discarded up to six times until antibodies were eliminated. Immunosuppressive induction with ATG was started for 5 days. Basic immunosuppression consisted of tacrolimus, mycophenolate mofetil, and prednisone. Extubation was performed on days 15, 2, and 1, respectively. After a follow-up of 17, 16, and 12 months all patients are well, ventricular function is excellent without any acute rejection periods; Patient 1 is still on dialysis. Isohemagglutinin titers against donor blood type have disappeared in follow-up. Conclusions: ABO-incompatible cardiac transplantation shows good short-term results in young infants and seems to be a safe procedure to lower the mortality on the waiting list. # 2007 European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V. All rights reserved. Keywords: Pediatric heart transplantation; ABO-incompatible; Congenital heart surgery; Heart transplantation 1. Introduction Donor organ shortage causes an estimated mortality 30 50% on the waiting list for pediatric heart transplant candidates of in Germany. Recently, West et al.  reported a series of successful ABO-incompatible heart transplants in newborns and young infants. They noted a remarkable decline in mortality rates on the waiting list from 58% to 10%. Blood-incompatible liver  and kidney  transplantation have been performed with favorable results, if antibody titers were reduced prior to transplantation. ABO-incompatible heart transplantation (Htx) other than with blood type O donors in a grown-up population has been done only by Presented at the joint 19th Annual Meeting of the European Association for Cardio-thoracic Surgery and the 13th Annual Meeting of the European Society of Thoracic Surgeons, Barcelona, Spain, September 25 28, * Corresponding author. Address: Cardiac Surgery, University Hospital Grosshadern, D Munich, Germany. Tel ; fax: address: (S.H. Daebritz). 1 Both authors contributed equally to the manuscript. mistake and is associated with a high mortality . This is in contrast to newborns and young infants, who have a relative immature immunologic system with limited production of isohemagglutinins. On the basis of this, West et al. started ABO-incompatible HTx in 1996, following a standardized protocol for preoperative antibody reduction by plasmapheresis . Comparable results have been reported by Rao et al. . Since December 2004, we transplanted three infants crossing the blood group compatibility barrier; during the same time, only one infant of the same age group received a donor organ compatible with the recipient s blood type. We report on our experience with these three patients. 2. Patients and methods All three patients had blood group O, isohemagglutinin titers were 1:4 or less, so that patients were amenable for ABO-incompatible heart transplantation according to the protocol defined by West et al. [West, personal communication]. Informed consent was given by the /$ see front matter # 2007 European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V. All rights reserved. doi: /j.ejcts
2 340 S.H. Daebritz et al. / European Journal of Cardio-thoracic Surgery 31 (2007) parents, the ethical committee of the German Physicians Association, and the Eurotransplant International Foundation Patient 1 The first patient was referred with hypoplastic left heart complex with aortic stenosis, ASD, and endocardiofibroelastosis. Interventional ASD-closure and aortic balloon valvuloplasty to establish two-ventricle circulation failed; left ventricular function did not improve and the left ventricle did not grow due to endocardfibroelastosis. Consecutively, the patient developed congestive heart failure (CHF) and renal failure necessitating peritoneal dialysis. After 2 months on the waiting list in high-urgent status, she was listed ABO-incompatible and received a donor organ blood type A1 2 weeks later, at the age of 7 months Patient 2 The second patient had restrictive hypertrophic cardiomyopathy with congestive heart failure and several periods of medical resuscitation. He was on inotropes and had to be ventilated on the waiting list. After 2 weeks in high-urgent status, he was listed ABO-incompatible because he became borderline for mechanical circulatory support. He received a donor organ blood type A1 1 week later, at the age of 5 months Patient 3 The third patient was diagnosed with dilative cardiomyopathy at the age of 2.5 months. She was directly listed in high-urgent status of ABO-incompatible being on inotropes, but not ventilated. After 1 day she received a donor organ blood type B at the age of 3.4 months Operative management ABO-incompatible transplantation in these patients was performed according to the protocol kindly provided by Lori West (formerly Hospital for Sick Children, Toronto). The parents gave informed consent for ABO-incompatible HTx. After permission of the Review Commission of the German Medical Council, Eurotransplant International Foundation agreed to allocate a respective donor organ under certain conditions. These included that no ABOcompatible recipient of the same size was waiting in highurgent status Pretransplant recipient management Anti-A1/A2 and anti-b antibody levels were determined in weekly intervals using standard reverse blood grouping hemagglutination methods described elsewhere . An urgent blood test was done just prior to transplantation. In addition, the blood bank provided a semiquantitative antibody measurement within 30 min ( quick-spin ), giving the results as negative or 1 4 positive . Intraoperatively, all blood samples were taken from the cardio-pulmonary bypass circuit (CPB). Patients 1 and 2 had positive antibody titers preoperatively. Although Patient 3 had only a 1+ positive semiquantitative assessment with negative titers, all patients underwent plasma exchange intraoperatively. The CPB circuit was filled with red blood cells (2 units) of blood type O (recipient blood type) and plasma (2 units) of the donor blood type (A or B in our patients) or AB. Red blood cells were irradiated and all blood products were CMV-negative (Table 1). Immediately after commencing CPB, whole blood was withdrawn via the venous line and volume was substituted from the CPB circuit via the arterial cannula (s.a.). During surgery, plasma exchange was done by withdrawal of 2 3- fold the patient s total blood volume. The red cell fraction from the extracted blood was separated, diluted, washed, and centrifuged achieving an elimination of plasma of 99% [6,7]. Antibodies were assessed at least twice after plasma exchange. In case antibodies were elevated, plasma exchange was repeated up to six times. Red cell retransfusion and donor organ reperfusion were not started until antibodies were negative or remained 1+ positive after several runs. Orthotopic heart transplantation was performed according to the standard technique of Lower and Shumway during plasma exchange Postoperative recipient management Postoperatively, quantitative antibody titers were assessed every day for 2 weeks and subsequently weekly for 2 months and after that monthly . After 6 months, patients were seen in 3-month intervals. Immunosuppression consisted of induction therapy with antithymocyte globulin (ATG; 3 mg/kg bolus, then 2 mg/kg/d), adjusted to reach a lymphocyte count of /ml until sufficient plasma levels of tacrolimus (Tac) were achieved. Intravenous methylprednisolone (30 mg/ kg) was given before release of the aortic cross-clamp, tapered down (3 mg/kg/d, 2 days, 2 mg/kg/d and Table 1 Anti-A1/A2 B antibody titers of Patients 1 and 2 Patient Recipient blood group Donor blood group Preoperative titers anti- 1-day postoperative anti- 1-week postoperative anti- Highest in follow-up Latest in follow-up A1 A2 B A1 A2 B A1 A2 B A1 A2 B A1 A2 B 1 O A 1:4 1:4 0 1: : :1 0 1: :1 2 O A 1: : :2 0 1: :1 3 O B (1+ a ) :1 0 1:1 1:1 1:1 1:1 1:1 1:1 0 a Quick-spin result.
3 S.H. Daebritz et al. / European Journal of Cardio-thoracic Surgery 31 (2007) mg/kg/d each for 2 days), and maintained with 0.5 mg/ kg/d. Maintenance immunosuppressants were Tac and mycophenolate mofetil (MMF) with through levels of and 2 5 ng/dl, respectively. Rejection monitoring included daily echocardiography, clinical signs, and cyto-immunological monitoring . After discharge, patients were seen monthly in the outpatient transplant clinic, for ECG, echocardiography, and control of their immunosuppressants. Cardiac catheterization or endomyocardial biopsies are not routinely performed in pediatric patients in our institution, but these patients are scheduled for biopsy in the second year after HTx. 3. Results All three patients were positive for antibodies, two had borderline titers (1:4) for anti-a/b according to the Toronto ABO-incompatible heart transplant protocol, and the third patient had 1+ positive assessment of anti-a and anti-b antibodies. Thus, all patients underwent plasma exchange on CPB. In Patient 1, after the fourth plasma exchange, the quick spin was 1+ and the corresponding anti-a titer was 1:1. As there was no change after another two exchange runs, these antibodies were accepted without any complication. Patient 2 had negative antibodies after five exchange runs. Patient 3 underwent one plasma exchange; thereafter antibodies were negative. Surgery, reperfusion, and weaning from CPB were uneventful in all three patients. Extubation was performed on postoperative days 15, 2, and 1, respectively. Patient 1 showed only a transient increase in urine output after Htx, so that she had to stay on peritoneal dialysis with intermittent periods of hemodialysis because of peritoneal catheter problems. Immunosuppression was administered according to the protocol (s.a.) except for Patient 1. She received additional monoclonal antibodies (daclizumab 6 mg/d) to cover low Tac levels, which were slowly increased as kidney function seemed to recover. However, she had to stay on peritoneal dialysis, causing a hospitalization of 3 months post-htx. Fig. 1. Anti-graft isohemagglutinins. Anti-A antibodies are at maximum 1:1, but anti-b titers are found at rising levels (Figs. 1 and 2) during follow-up of 17 months. The patient is doing well apart from the peritoneal dialysis performed at home. She has undergone cardiac catheterization with biopsy without any pathological finding. In Patient 2, anti-a1 levels became detectable on day 3 (titer 1:1) without any obvious impairment of hemodynamic function; titers disappeared after 3 months. The patient is in excellent clinical condition after a follow-up of 16 months. He has developed low titers against blood type B (Figs. 1 and 2). In Patient 3, anti-b antibodies were detected after 1 month, but then disappeared. After a follow-up of 12 months the clinical course has been uneventful. She has no anti-b titers, but low titers against A1 and A2 (Figs. 1 and 2). Overall, titers against the respective donor blood type are not found while isohemagglutinins against the remaining blood types develop at low levels. The clinical course is uneventful without any rejection episodes and normal ventricular function in echocardiography. 4. Discussion Fig. 2. Anti-non graft isohemagglutinins (log scale). Pediatric heart transplant recipients on the waiting list have a higher mortality compared to adults . First, the donor and recipient pool is much smaller, so that some organs cannot be allocated due to the unavailability of a suitable recipient despite donor shortage. Second, bridging methods are limited and carry a mortality of up to 50% . On the basis of the increased knowledge of the immaturity of the immune system of the newborns and young infant, particularly with respect to carbohydrate antibody production, West et al. introduced and successfully performed ABO-incompatible heart transplantation in this age group . In adults, ABO-incompatible liver and kidney transplantation has shown good results [3,11,12]. In Japan living related kidney transplantation with preoperative antibody removal by double filtration plasmapheresis (DFPP) was evaluated . Long-term graft survival was 76.9 and 55.9% after 5 and 10 years and independent of preoperative anti-a/b antibody titers . However, in cadaveric kidney transplantation of ABO-incompatible grafts, the 1- and 10-year survival rates of
4 342 S.H. Daebritz et al. / European Journal of Cardio-thoracic Surgery 31 (2007) and 56% still remain inferior compared with 96 and 67% of ABO-compatible grafts . In contrast, heart transplantation crossing blood type barriers has only been done sporadically and mostly result from erroneous allocation of donor organs . In a survey of almost 5000 heart transplants, Cooper identified only eight ABO-incompatible heart transplants ; five hearts were hyperacutely rejected, three patients had retransplantation, and early mortality was 50%. In other case reports urgent retransplantation with ABO-compatible grafts was performed [14,15]. Only sporadically, ABO-incompatible thoracic transplantation showed good outcome under use of additional immunosuppressants (ATG, anti-cd20, TP10), C1-esterase inhibitors, plasmapheresis, and exchange and extracorporeal immunoadsorption [16 18]. Therefore, ABO-incompatible heart transplantation is not recommended in adults . Due to the immature immune system in small children which starts to mature by contact with gut bacteria after birth, isohemagglutinins are undetectable during the first months of life . Thus, in 2001, West et al. reported a series of 10 infants less than 14 months of age who underwent ABO-incompatible heart transplantation in Toronto . During CPB, the total body volume of recipient plasma was exchanged until circulating blood type antibodies were undetectable. No hyperacute rejection occurred; overall survival rate was 80%. Two deaths 24 and 29 days after transplant were unrelated to ABO incompatibility. A followup 3 years later revealed donor-specific B-cell tolerance in the recipients . B-cells were capable of producing antibodies other than those against the donor blood type, so that the lack of donor-specific antibodies was not due to immunosuppression or dysfunction of B-cells. There is now evidence that donor-specific B-cell elimination is the mechanism of tolerance induction in recipients of ABOincompatible donor hearts . All three recipients in our study had blood type O associated with a longer waiting time and mortality on the waiting list . Thus, we started a program of ABOincompatible heart transplantation in infants combining intraoperative plasma exchange during CPB with our standard immunosuppressive protocol. The standard agglutination tests to determine anti-a and -B antibodies were complemented by the quick spin isohemagglutinin test which was established at our Department of Transfusion Medicine with a 24 h availability. In one of our patients, an anti-a1 titer of 1:1 remained detectable after six plasma exchange runs without any hyperacute or delayed rejection. Obviously, a remaining antibody titer of 1+ in quick spin is acceptable. The autotransfusion device used perioperatively to recover autologous red cells proved to be safe to remove all circulating isohemagglutinins during the washing procedure. Isohemagglutinin titers were undetectable in all samples that were taken from washed red cells (data not shown). The upper limit of the preoperative isohemagglutinin antibody titer and/or age for ABO-incompatible heart transplantation remains to be determined. West et al. successfully transplanted one 14-month-old child with a 1:128 anti-a titer while Rao et al. did not exceed a maximum titer of 1:8, although the oldest recipient was 21 months old. Our recipients had maximum titers of 1:4 and were under 1 year of age. In summary, isohemagglutinin titers seem to be better predictors of postoperative outcome than age. Donor-specific isohemagglutinins disappeared in all three of our patients within 4 months while the corresponding antinon-graft isohemagglutinins remained detectable at low levels. These results reflect the experience of the Toronto group that infants develop acquired B-cell tolerance most probably due to donor-specific B-cell elimination [20,21]. Our immunosuppressive protocol as well as that of the Toronto group included Tac and MMF, because MMF is supposed to have a greater effect on B-lymphocytes and thus on antibody production . Shimmura et al. reported favorable results of ABO-incompatible living kidney transplantation in adults under Tac or MMF in comparison to cyclosporine and azathioprin . The use of Tac and MMF may therefore have contributed to the excellent outcome even in the subset of patients beyond the neonatal period [21,22]. In conclusion, our results reconfirm that ABO-incompatible heart transplantation in the infant age group is safe provided that preoperative isohemagglutinin titers are low and intraoperative plasma exchange is performed until titers are almost undetectable. As the outcome after transplantation is not adversely altered  we are encouraged to proceed with the program in order to reduce mortality on the waiting list as shown by the Toronto group and others [5,22 24]. References  West LJ, Pollock-Barziv SM, Dipchand, Lee KJ, Cardella CJ, Benson LN, Rebeyka IM, Coles JG. ABO-incompatible heart transplantation in infants. N Engl J Med 2001;344:  Wu A, Bühler LH, Cooper DKC. ABO-incompatible organ and bone marrow transplantation: current status. Transpl Int 2003;16:  Ishida H, Koyama I, Sawada T, Utsumi K, Murakami T, Sannomiya A, Tsuji K, Yoshimura N, Tojimbara T, Nakajima I, Tanabe K, Yamaguchi Y, Fuchinoue S, Takahashi K, Teraoka S, Ito K, Toma H, Agishi T. Anti-AB titer changes in patients with ABO incompatibility after living related kidney transplantations: survey of 101 cases to determine whether splenectomies are necessary for successful transplantation. Transplantation 2000;70:  Cooper DKC. 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5 S.H. Daebritz et al. / European Journal of Cardio-thoracic Surgery 31 (2007)  Alexandre GP, Squifflet JP, De Bruyere M, Latinne D, Reding R, Gianello P, Carlier M, Pirson Y. Present experiences in a series of 26 ABO-incompatible living donor renal allografts. Trans Proc 1987;19:  Tanabe K, Tokumoto T, Ishida H, Toma H, Nakajima I, Fuchinoue S, Teraoka S. ABO-incompatible renal transplantation at Tokyo Women s Medical University. Clin Transpl 2003;  Shimmura H, Tanabe K, Ishida H, Tokumoto T, Ishikawa N, Miyamoto N, Shirakawa H, Setoguchi K, Nakajima I, Fuchinoue S, Teraoka S, Toma H. Lack of correlation between results of ABO-incompatible living kidney transplantation and anti-abo blood type antibody titers under our current immunosuppression. Transplantation 2005;80:  Pikul FJ, Bolman RM, Saffitz JE. Anti-B-mediated rejection of an ABOincompatible cardiac allograft despite aggressive plasma exchange transfusion. Trans Proc 1987;19:  Albrechtsen D, Geiran O, Foerster A, Fauchald P, Flesland O, Lindberg H, Solheim BG, Vartdal F, Froysaker T. Cardiac transplantation from a blood group ABO-incompatible donor: a case report. Trans Proc 1990;22:143.  Banner NR, Rose ML, Cummins D, de Silva M, Pottle A, Lyster H, Doyle P, Carby M, Khaghani A. Management of an ABO-incompatible lung transplant. Am J Transplant 2004;4(7):  Koestner SC, Kappeler A, Schaffner T, Carrel TP, Nydegger UE, Mohasci P. Histo-blood group type change of the graft from B to O after SBO mismatched heart transplantation. Lancet 2004;363:  Pierson RN, Loyd JE, Goodwin A, Majors D, Dummer JS, Mohasci P, Wheeler A, Bovin N, Miller GG, Olson S, Johnson J, Rieben R, Azimzageh A. Successful management of an ABO-mismatched lung allograft using antigen-specific immunoadsorption, complement inhibition, and immunomodulatory therapy. Transplantation 2002;74(1):  Fong SW, Qaqundah BY, Taylor WF. Developmental patterns of ABO isohemagglutinins in normal children correlated with the effects of age, sex, and maternal isohemagglutinins. Transfusion 1974;14:  Fan X, Ang A, Pollock-Barziv SM, Dipchand AI, Ruiz P, Wilson G, Platt JL, West LJ. Donor specific B-cell tolerance after ABO-incompatible infant heart transplantation. Nat Med 2004;10:  West LJ. B-cell tolerance following ABO-incompatible infant heart transplantation. Transplantation 2006;81:  West LJ, Karamlou T, Dipchand AI, Pollock-BarZiv SM, Coles JG, McCrindle BW. Impact on outcomes after listing and transplantation, of a strategy to accept ABO blood group-incompatible donor hearts for neonates and infants. J Thorac Cardiovasc Surg 2006;131:  McMahon AM, van Doorn C, Burch M, Whitmore P, Neligan S, Rees P, Radley-Smith R, Goldman A, Brown K, Cohen G, Tsang V, Elliott M, de Leval MR. Improved early outcome for end-stage dilated cardiomyopathy in children. J Thorac Cardiovasc Surg 2003;126:  Rodriguez RJ, Addonizio LJ, Lamour JM, Mital S, Mosca R, West LJ, Nova JC, Hsu DT. Pediatric heart transplantation across ABO blood type barriers: a case study. Prog Transplant 2005;15: Appendix A. Conference discussion Dr S. Aharinejad (Vienna, Austria): Do you know anything about the longterm survival, overall survival, not from your studies but published in the literature? I m not in this field. It s just for my curiosity. Dr Daebritz: The longest follow-up is 8 years now in the registry. It is from the Toronto group, and, as they are running the registry, they compared ABOincompatible to ABO-compatible results. The ABO-incompatible patients are even slightly better, but this, of course, is not statistically significant. Dr S. Brann (St. Augustine, Trinidad and Tobago): Can you please tell me how your postop immunosuppression has changed, if at all? Dr Daebritz: It hasn t changed at all. We use the standard immunosuppression regimen, which is used for ABO-compatible transplantation as well. Dr C. Stremmel (Freiburg, Germany): I have a question concerning the long-run. In the literature, do these transplanted people need less immunosuppressive agents after 1 or 2 years so that they are also tolerant on the T-cell field? Dr Daebritz: There is no evidence on that. We don t have any idea what this exposure to another blood type does with the T-cell immune system. It may sensitize it, but it may also cause tolerance. This is totally unknown and both are possible. Therefore, nobody has so far reduced immunosuppression in these patients.