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(Circulation. 1996;94:1063-1067.)
© 1996 American Heart Association, Inc.

Articles

Prospective Analysis of HLA Immunogenicity of Cryopreserved Valved Allografts Used in Pediatric Heart Surgery

Robert E. Shaddy, MD; Dixie D. Hunter, RN; Karen A. Osborn, RN; Linda M. Lambert, BS; L. LuAnn Minich, MD; John A. Hawkins, MD; Edwin C. McGough, MD; Thomas C. Fuller, PhD

the Departments of Pediatrics (R.E.S., D.D.H., K.A.O., L.L.M.), Surgery (L.M.L., J.A.H., E.C.M.), and Pathology (T.C.F.), University of Utah School of Medicine and Primary Children's Medical Center (Salt Lake City).

Correspondence to Robert E. Shaddy, MD, Division of Cardiology, Primary Children's Medical Center, 100 N Medical Dr, Salt Lake City, UT 84113. E-mail shaddy@med.utah.edu.

	Abstract

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Background The HLA immunogenicity of cryopreserved valved allografts used in the surgical repair of congenital heart defects is unknown.

Methods and Results To determine the immunogenicity of these allografts, we measured prospectively the frequency of panel-reactive HLA class I alloantibodies (PRA) before, 1 month after, and 3 months after allograft implantation in 9 children (age, 5.4±2.1 years) and after open-heart surgery without allograft implantation in 11 age-matched control children (age, 4.0±1.5 years). PRA was determined against an HLA-select frozen T-lymphocyte panel using the antiglobulin cytotoxicity technique. After allograft implantation, PRA increased from 3.2±2.7% before surgery to 63.3±12% at 25±2 days after surgery and 99.7±0.3% at 3.4±0.3 months after surgery. The use of dithiothreitol to remove IgM alloantibodies resulted in a modest decrease in PRA at 1 month (33.2±13%) but no change at 3 months (93.0±3.4%), suggesting the initial humoral response is an IgM alloantibody that switches almost exclusively to IgG by 3 months. Control patients showed no increase in PRA over time: 1.6±1% before surgery, 3.2±1% at 28±5 days after surgery, and 1.7±1% at 2.7±0.3 months after surgery.

Conclusions Cryopreserved valved allografts in children induce a marked HLA alloantibody response that increases to broad panel reactivity within 3 months after surgery. This HLA sensitization has potential not only for causing deleterious effects on allograft function but also for limiting the future opportunity of heart transplantation in patients who receive cryopreserved valved allografts.

Key Words: heart defects, congenital • immune system • pediatrics • valves

	Introduction

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Valved allografts are commonly used in both the pulmonary and aortic positions for the surgical repair of congenital heart defects. Although the freedom from reoperation in these patients has been reported to approach 80% to 90% in the intermediate term,^{1 2 3} there continue to be problems with allograft dysfunction manifested as allograft valve stenosis and insufficiency, allograft calcification, and allograft luminal narrowing.^{1 3 4 5} Risk factors for allograft calcification and dysfunction include young age^{3 4 6} and the type of allograft: aortic rather than pulmonary allograft.^{1 2 3 4} The role of immune-mediated injury to the allograft has been a matter of controversy for decades, and initial studies failed to demonstrate evidence of an immune reaction sufficiently severe to be clinically important.^{7 8} However, a donor-specific alloantibody response to MHC class I antigens has been demonstrated in experimental allografts in rats and in fresh venous allografts in dogs.^{9 10 11} More recently, adults have been found to generate a strong donor HLA-specific antibody response to aortic valve homovital and antibiotic-sterilized homografts.¹² Whether cryopreservation of allografts abrogates their immunogenicity is controversial.^{13 14 15} The purpose of the present study was to examine prospectively the humoral immune response of children receiving cryopreserved valved allografts as part of their repair for congenital heart disease.

	Methods

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Nine patients who received cryopreserved valved allografts for congenital heart disease surgery were enrolled in the study. The patients were from 1 week to 17 years old (mean±SEM age, 5.4±2.1 years). Diagnoses were truncus arteriosus in four patients, congenital aortic stenosis and regurgitation in three patients, tetralogy of Fallot in one patient, and valvar pulmonic stenosis in one patient. Eleven children undergoing intracardiac surgical repair of congenital heart disease without placement of a valved allograft were enrolled in the study as age-matched controls. These patients were from 1 week to 13 years old (mean±SEM age, 4.0±1.5 years). Diagnoses were ventricular septal defect in four patients, atrial septal defect in four patients, and total anomalous pulmonary venous return, aortic stenosis, and pulmonary atresia with intact ventricular septum in one patient each. Five patients in the allograft group and three patients in the control group had undergone previous heart surgery; none of them had had allografts placed previously.

The Institutional Review Board of Primary Children's Medical Center approved the study protocol, and informed consent was obtained from a parent or guardian of each patient before entry into the study. Blood for panel-reactive antibody (PRA) determination was obtained from each patient at the following times: immediately before the initiation of cardiopulmonary bypass, 1 month after surgery, and 3 months after surgery. Blood products received by all patients were irradiated and leukocyte filtered before administration to remove allogeneic white blood cells that could sensitize the patients. Blood products were irradiated with ¹³⁷Cs at 30 Gy and were filtered with the use of Sepacell leukocyte removal filters (Fenwal Division, Baxter Healthcare Corp). All information regarding allograft type, size, ABO blood type, and HLA phenotype was obtained, when available, from the providing company (Cryolife, Inc).

The type of cryopreserved valved allograft inserted into the patients was an aortic allograft in four patients and a pulmonary allograft in five patients. The allograft was placed between the right ventricle and the pulmonary artery in all patients; the patients with aortic stenosis and regurgitation had a pulmonary autograft placed in the aortic position and a cryopreserved valved allograft in the orthotopic pulmonary position. The size of the allograft was 15.7±1.4 mm in diameter (range, 10 to 20 mm). The ages of the allograft donors were very similar to the ages of the children who received the allografts: 5.9±1.5 years. The length of time that the valved allografts were cryopreserved was 7.5±2.8 months (range, 1.1 to 25.2 months). In the patients who received allografts, five patients were blood type A and four patients were blood type O. The donor blood type for the valved allograft was available from the company for only four allografts, and only one of these four allograft/recipient blood type matches was ABO incompatible (blood type B allograft into blood type O patient). The HLA phenotype for the valved allografts was available for only three donors.

HLA-A, -B, and -C loci serotyping was performed on all patients using the standard complement-dependent cytotoxicity (CDC) test¹⁶ and in-house serological reagents. PRA was determined using the sensitive anti-human light-chain immunoglobulin (AHG)-CDC technique¹⁷ against a frozen T-lymphocyte panel composed of 40 individuals of diverse HLA type and racial background.¹⁸ Briefly, 0.001 mL of each patient's serum was incubated with 0.001 mL of cells (4x10⁶/mL) for 30 minutes. After being washed four times with 0.005 mL of culture medium, 0.001 mL of an optimal dilution of AHG (One Lambda, Inc) was added. After 2 minutes, 0.005 mL of rabbit complement (1:2 dilution) was dispensed, and the results of cytotoxicity were read microscopically after 60 minutes with the use of ethidium bromide dye exclusion. PRA was expressed as the percentage of lymphocyte panel members against which the patient's serum reacts and thus against which the patient has HLA class I antibody.

In several experiments, PRA positive sera were adsorbed with pooled platelets to confirm the HLA class I specificity of the cytotoxic sera.¹⁷ To differentiate IgM from IgG HLA antibody, 0.195 mL of serum was incubated with 0.005 mL of 0.2 mol/L dithiothreitol (Sigma Chemical Co) in phosphate-buffered saline for 30 minutes at 37°C.¹⁹ The serum, now free of IgM antibody, was then screened through AHG-CDC as described above.

Statistical Analysis
Comparisons between continuous data were made with ANOVA and Scheffe's post-hoc analysis. Comparisons between nominal data were made with Fisher's exact test. Correlations were performed with Pearson's correlation coefficient. Differences were considered significant for a value of P<.05. All data are expressed as mean±SEM.

	Results

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In patients who received a cryopreserved valved allograft, PRA increased from 3.2±2.7% before surgery to 63.3±12% at 25±2 days after surgery and 99.7±0.3% at 3.4±0.3 months after surgery (P<.01) (Figure). To investigate further the nature of the humoral response, studies were undertaken to determine the Ig class, titer, and specificity of the antibodies induced from implantation of these cryopreserved valved allografts. Use of dithiothreitol to remove IgM resulted in a modest decrease in PRA at 1 month (to 33.2±13%) but no change in PRA at 3 months (93.0±3.4%), suggesting that the primary humoral response is IgM that was detected at a time point (1 month) when the B-cell response was switching to IgG. By 3 months, PRA was found to be high-titered IgG and specific against HLA class I alloantigens. There were no differences in the PRA between aortic and pulmonary allografts. There was no correlation between age of the patient and PRA; all patients developed a PRA of >95% within 3 months of surgery. Control patients showed no significant increase in PRA over time: 1.6±1% before surgery, 3.2±1% at 28±5 days after surgery, and 1.7±1.2% at 2.7±0.3 months after surgery.

View larger version (16K): [in this window] [in a new window]   Figure 1. Bar graph showing panel-reactive antibody in allograft patients (patients receiving an allograft) and control patients (patients not receiving an allograft) before, 1 month after, and 3 months after surgery. *P<.01 compared with control patients and compared with allograft patients before surgery.

The initial response generated in all patients who received an allograft was predominantly a dithiothreitol-sensitive, IgM antibody that rapidly switched to IgG PRA within 3 months of surgery (Table). Since platelets express reasonable levels of HLA class I alloantigens, sera from four patients, collected 3 months after surgery, were adsorbed twice with equal volumes of pooled, washed platelets. In all cases, the PRA titers dropped significantly, although reactivity was still present in two cases. However, on titration of these sera, as well as sera from several other patients, it was clear that the actual titers of the HLA alloantibodies were exceedingly high, often with end points of >1:256. Determination of the actual HLA specificity of the alloantibodies was quite difficult because of the broad pattern of panel reactivity. However, in the sera that were collected 1 month after allografting, there were clearly defined HLA antibody specificities that were consistent with the HLA phenotypes of the patients; that is, the PRA was directed against major alloantigen groups that were not expressed by the antibody responders. Finally, development of HLA class II alloantibody, although a definite possibility considering the brisk response against HLA class I, unfortunately could not be determined because of limitations of serum volume in these children.

View this table: [in this window] [in a new window]   Table 1. Individual PRA Data on Patients Who Received Valved Allografts

The number of patients who were given perioperative blood products was not significantly different between patients who received allografts and control patients. Five patients who received allografts received whole blood while undergoing cardiopulmonary bypass; four of the allograft patients had cardiopulmonary bypass without blood products. However, a total of eight of the nine patients (88%) who received allografts were transfused with either packed red blood cells, whole blood, or platelets after the surgery because of excessive blood loss. In the control group, five patients received whole blood at the time they underwent cardiopulmonary bypass, and a total of six patients (55%) received blood products perioperatively.

	Discussion

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Results of the present study demonstrate clearly that children who undergo surgical repair of congenital heart defects in which a cryopreserved valved allograft is used develop an HLA humoral antibody response within 1 month after surgery that continues to broaden in reactivity to 3 months after surgery. These data are in agreement with experimental data from research in animals that have shown increased levels of donor-specific anti–T-cell antibodies in valve and skin allografts in rats¹⁰ and in vitro experimental data from research in humans that have shown that human cardiac valves were able to stimulate an immune response in lymphocyte culture.¹³ Smith et al¹² recently demonstrated a strong HLA-specific alloantibody response in adults who underwent aortic valve replacement with either a homovital allograft, an antibiotic-sterilized allograft, or a porcine xenograft. This antibody response was more prevalent, stronger, and longer lasting in the homovital allograft recipients than in the antibiotic-sterilized allograft recipients. Homovital allografts were used within 7 days of explantation, and antibiotic-sterilized allografts were stored at 4°C for 2 months. This decreased antibody response in the older grafts suggested a possible abrogation of the antibody response with time.

Since the average time of storage of the cryopreserved allografts used in our study was 7.5 months, we believe that neither time nor cryopreservation effectively eliminates the immunogenicity of these allografts in children. Other studies support this; although cryopreservation may result in some loss of viable endothelium in human allografts,²⁰ cryopreservation does not alter the antigenicity of aortic allografts in mice²¹ or of osteoarticular allografts in humans.²² Our data also demonstrate that the type of allograft (aortic versus pulmonary) does not alter the antibody response, since both types of allografts were associated with similar PRAs. There has been speculation that an increased proportion of CD5+ B lymphocytes in young children may be at least partially responsible for an increased incidence of allograft dysfunction in younger children compared with older children.⁴ Our data indicate that the humoral HLA alloantibody response is quite marked in all children, regardless of age.

There were no differences between groups with regard to the number of patients who were given blood products during cardiopulmonary bypass. However, there was a tendency for more children in the allograft group to receive blood products (88%) around the time of surgery than those in the control group (55%). All blood products in both groups were irradiated and leukocyte filtered; this should remove the majority of the white blood cell stimulus and thus reduce risk of sensitization to HLA. Furthermore, none of the five control patients who received perioperative blood products developed an antibody response. Thus, the facts that none of the patients in the control group developed HLA antibodies and that all of the patients in the allograft group developed a marked response make it virtually certain that the etiology of the HLA alloantibody response is the presence of the allograft rather than blood products or some other aspect of the cardiopulmonary bypass or surgical procedure.

The short length of follow-up in the present study does not allow for analysis of the duration of the immune response to the allograft. However, the sustained nature of the antigenic stimulus provided by an implanted allograft would lead one to expect a prolonged and broad elevation in HLA alloantibodies. Viability of the implanted allograft would provide a constant immune stimulus against which the host could respond. Broadening of the alloantibody response is a common occurrence in patients with end-stage renal disease who require multiple transfusions.²³ Also, in renal allograft recipients who are in a chronic phase of rejection, the HLA class I alloantibodies become broad and are directed against the high-frequency HLA class I public epitopes rather than the narrow, private specificities that are unique to each individual HLA allele.^{23 24} In contrast, with transfused patients, rarely do >30% produce HLA alloantibody, which, in many instances, is transient and of low titer (T.C. Fuller, personal communication). Adult patients have also been found to have HLA alloantibodies as long as 15 years after allograft implantation, although it is not certain that the allograft is responsible for the persistent alloantibody response.¹² In addition, part of the impetus for us to perform this study was the incidental finding of HLA alloantibodies in three children at our institution who had previously undergone surgical repair of a congenital heart lesion with a cryopreserved valved allograft. These three children were found to have markedly elevated PRAs between 2 and 3 years after their initial allograft surgery when they were evaluated for heart transplantation.

The hemodynamic consequences of this antibody response on long-term graft function are unknown. In dogs, histoincompatible femoral vein allografts thrombosed, whereas autografts remained patent.¹¹ Another possible manifestation of immune recognition of a valved allograft may be calcification. Experimental rat heterotopic valved allografts show significantly more calcification than do syngeneic grafts, although the degree of allogeneicity does not appear to influence the magnitude of calcification.²⁵ However, it is unclear as to which effector arm of the immune response is responsible for this immunological process of graft calcification and dysfunction. Previous xenograft studies in mice have demonstrated no differences in the degree of calcification of subcutaneously implanted porcine valve cusps into normal and athymic mice,²⁶ suggesting that T cells are not necessary for calcification of valve xenografts. The role of ABO blood type mismatch is also unknown. Retrospective analysis of a group of children from this institution failed to show a correlation between allograft calcification and ABO incompatibility.¹ Therefore, although it is likely that immunological recognition of allografts contributes to allograft calcification and/or dysfunction, the extent and mechanisms of this association are still unclear. Whether immunosuppression will decrease HLA sensitization and/or allograft dysfunction remains to be seen. There obviously are other factors involved in allograft dysfunction and calcification, since it appears that HLA sensitization occurs in a very high percentage of patients, yet only a small proportion develop allograft dysfunction or radiographic evidence of calcification in the intermediate term.² Experimental evidence in rats suggests that cyclosporine alters intimal thickening, aneurysm formation, and humoral response of allografts.^{27 28} Further studies in humans are indicated to determine the long-term risks and benefits of immunosuppression in this setting.

The development of anti-HLA antibodies poses a significant problem for any patient who may require heart transplantation at a later date. Allografts (valved or nonvalved) are used in a large variety of congenital heart disease operations, including hypoplastic left heart syndrome, truncus arteriosus, D-transposition of the great arteries with ventricular septal defect and pulmonary stenosis, reconstruction of branch pulmonary arteries, and aortic or pulmonary stenosis and regurgitation.^{1 29} Because of the concern that circulating donor-specific HLA alloantibodies decrease graft survival,^{30 31} patients with an elevated PRA before transplantation often require a prospective crossmatch between donor and recipient before acceptance of a donor organ. This can significantly limit the donor pool available to an otherwise medically suitable recipient, a problem that only magnifies the already significant donor shortage present in organ transplantation. In addition, there is evidence that patients with elevated PRAs have a higher degree of allograft loss and a higher posttransplantation mortality, even in the presence of a negative lymphocyte crossmatch.^{32 33} Thus, use of a cryopreserved allograft in a patient who subsequently requires heart transplantation not only may limit donor availability but also may limit graft survival. Further study is needed to determine whether HLA typing of valved allografts may be beneficial or even feasible in congenital heart disease surgery.

In conclusion, cryopreserved valved allografts induce a marked HLA-specific IgG alloantibody response that increases to broad panel reactivity within 3 months of surgery. This HLA sensitization has potential not only for causing deleterious effects on allograft function but also for limiting the future opportunity of heart transplantation in patients who receive allografts.

	Acknowledgments

 
This investigation was supported by US Public Health Services research grant MO1-RR-00064 from the National Center for Research Resources. We thank Tracie Profaizer for her technical assistance with HLA serology and Kris Sjoblom for her expert editorial assistance.

Received December 8, 1995; revision received March 1, 1996; accepted March 4, 1996.

	References

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