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Ann Thorac Surg 2005;79:641-644
© 2005 The Society of Thoracic Surgeons

Original article: Cardiovascular

Panel-Reactive Antibodies Late After Allograft Implantation in Children

David K. Hooper, MDa, John A. Hawkins, MDb, Thomas C. Fuller, PhDc, Tracie Profaizer, BSc, Robert E. Shaddy, MDa,*

a Department of Pediatrics
b Department of Surgery
c Department of Pathology, University of Utah School of Medicine and Primary Children's Medical Center, Salt Lake City, Utah

Accepted for publication July 22, 2004.

* Address reprint requests to Dr Shaddy, Cardiology, Suite 1500, Primary Children's Medical Center, 100 N Medical Dr, Salt Lake City, UT 84113 (E-mail: robert.shaddy{at}ihc.com).


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Comment
 References
 
BACKGROUND: Circulating human leukocyte antigen (HLA) panel-reactive antibodies (PRA > 10%) have been independently associated with increased risk of rejection and mortality in patients who undergo cardiac transplantation. Cryopreserved allografts used to repair heart defects induce broadly reactive HLA antibodies in children that persist for an undetermined duration of time. The purpose of this study was to prospectively determine the level of HLA sensitization several years after implantation of cryopreserved allografts in children.

METHODS: We conducted late follow-up of 13 children previously screened for PRA before and after implantation of valved and nonvalved allografts who are alive and free from allograft replacement. Panel-reactive antibodies against HLA class I and II antigens were determined using flow cytometry and classified as high reactive (>50% PRA), low reactive (11% to 50%), or absent (0% to 10%). Follow-up PRA was compared with PRA obtained 3 months after initial allograft implantation.

RESULTS: Elevated HLA class I PRA persisted at late follow-up in 12 of 13 children, although it decreased significantly from high to low or from low to absent in 12 of 13 patients (p < 0.001). Elevated HLA class II PRA persisted at late follow-up in 6 of 13 children (46%) and had decreased significantly from prior levels (p = 0.011).

CONCLUSIONS: Circulating HLA antibodies induced by cryopreserved allograft tissue persist up to 8 years after implantation although they decrease with time. Therefore, children who have received cryopreserved allografts before cardiac transplantation may be at greater risk for transplant rejection.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Comment
 References
 
Cryopreserved valved allografts and nonvalved allograft patches are used widely in the surgical repair of a number of congenital heart defects. Cryopreserved aortic and pulmonic valves are used to reconstruct the right and left ventricular outflow tracts, whereas cryopreserved allograft patches are used in the reconstruction of the aortic arch and pulmonary arteries. Unlike solid organ transplants, cryopreserved allografts are not typically human leukocyte antigen (HLA) typed or crossmatched with their donor before implantation. During the past several years, substantial evidence has accumulated that recipients mount both cellular and humoral immune responses against donor-specific HLA class I and II antigens on implanted cryopreserved allografts [1–8]. This antibody response has broad HLA specificity, reaches a panel-reactive antibody (PRA) frequency of nearly 100% by 3 months after implantation, and persists for at least 1 year [2, 9]. Yet, although retrospective review has suggested that PRA may persist for up to 15 years after implantation of cryopreserved allografts in adults [1], the long-term persistence of circulating antibodies after implantation of cryopreserved valved and nonvalved allografts in children has never been demonstrated prospectively.

The effects of persisting, elevated PRA on the long-term function of allografts are unknown, although it has been suggested that they may contribute to premature valve failure in children and adults [3, 4, 8, 10, 11]. The persistence of circulating HLA antibodies also holds significance for the small number of children who, at a young age, require palliative heart procedures and who later go on to require heart transplantation. Because patients with an elevated PRA before heart transplantation may be at increased risk of antibody-mediated, acute accelerated transplant rejection [12–14], it is common practice to subject these patients to prospective donor-specific crossmatches before transplantation. This practice greatly decreases the available donor pool for each recipient and can result in increased waiting time, morbidity, and mortality. Furthermore, even in the setting of a negative donor-specific crossmatch, presensitization to HLA is independently associated with increased risk of rejection and mortality in adults [14].

We hypothesized that PRA persist for several years after the implantation of cryopreserved allografts in children. Therefore the purpose of this present study was to prospectively determine the level of PRA sensitization in children several years after the implantation of cryopreserved valved and nonvalved allografts used in the repair of congenital heart defects.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Comment
 References
 
Study Design
The Institutional Review Board at Primary Children's Medical Center and the University of Utah approved this study. We conducted late follow-up (median, 4.6 years; range, 3.6 to 8.4 years) of 35 children who were consecutively enrolled between August 1994 and October 1999 in previous studies to determine PRA before and after the implantation of cryopreserved valved and nonvalved allografts to repair congenital heart defects. These children had exhibited an almost undetectable PRA before surgery and a broad PRA response to HLA class I and II antigens within 3 months of implantation of the allograft [2, 6, 15]. All subjects who were alive and free from allograft replacement since their original study procedure were approached for enrollment in our current study. Subjects who had received additional allograft material since the time of the original studies were excluded because of the confounding effect that the new allograft would have. Blood samples for follow-up PRA analysis were collected from each patient who gave consent. Data regarding patient age at original operation, diagnosis, treatment, and type of allograft were obtained from the records of the prior studies.

Patients
Sixteen children were alive and free from allograft replacement at the time of follow-up; however, 3 of these patients refused participation in our study. Thus, 13 children were enrolled in our follow-up study. Age at original implantation of the allograft in these study subjects ranged from 6 weeks to 10.4 years (median, 1.1 year), and time of follow-up ranged from 3.6 years to 7.9 years (median, 4.6 years.) Of the children enrolled in follow-up, 4 had received pulmonary artery patch allografts, 3 had received pulmonary artery monocusp patch allografts, and 6 had received pulmonary valve allografts. Diagnoses and treatments for these children are listed in Table 1.


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  		Table 1. Diagnoses and Treatments in the 13 Patients
 
Panel-Reactive Antibody Analysis
Panel-reactive antibody analysis was performed in the Histocompatibility and Immunogenetics Laboratory at the University of Utah according to a previously published procedure [16]. Panel-reactive antibody is expressed as the percentage of lymphocyte panel members against which each patient's serum reacts and therefore reflects the breadth of allosensitization against the potential donor population. We classified the PRA of each child as follows: a PRA of less than or equal to 10% was considered nonreactive, PRA of 11% to 50% was considered low reactive, and PRA in excess of 50% was considered high reactive. Follow-up PRA from this study was compared with PRA obtained at 3 months after the implantation of allograft tissue. Statistical analysis was performed using a Fisher's exact test comparing number of patients with high reactive PRA to the number of patients with either low reactive or nonreactive PRA at 3 months and at late follow-up.


   Results
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 Abstract
 Introduction
 Patients and Methods
 Results
Comment
 References
 
Human leukocyte antigen class I PRA was nonreactive in all children before allograft implantation and was high reactive in 12 of 13 children and low reactive in 1 child at 3 months after implantation (previously published results) [2, 5, 6, 15]. At late follow-up elevated HLA class I PRA persisted in 12 of 13 children. However, in 10 of these children PRA decreased from high reactivity to low reactivity, and it remained highly reactive in only 2 children (p < 0.001). In the child with low reactive PRA at 3 months, PRA decreased to nonreactive by the time of follow-up.

Human leukocyte antigen class II PRA was nonreactive in all children before allograft implantation and at 3 months was highly reactive in 8 of 13 children, low reactive in 4 children, and nonreactive in 1 child (previously published results) [5, 6, 15]. At late follow-up elevated HLA class II PRA persisted in only 6 children. Panel-reactive antibody was high reactive in 1 child, low reactive in 5 children, and nonreactive in the remaining 7 children (p = 0.011). Human leukocyte antigen class I and II PRA are depicted in Figure 1.



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  		Fig 1. Human leukocyte antigen (HLA) class I (top) and II (bottom) panel-reactive antibody (PRA) of each patient who received a cryopreserved allograft. Panel-reactive antibody was assessed before implantation of the allograft, 3 months after implantation, and at long-term follow-up (3.6 to 7.9 years). We compared high reactive PRA (PRA > 50%) to low or nonreactive PRA (PRA < 50%) at 3 months and at follow-up. Decrease in PRA was statistically significant for both human leukocyte antigen class I (p < 0.001) and human leukocyte antigen class II (p = 0.011) antibodies.

 

   Comment
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Comment
References
 
In this prospective study we found that the broadly reactive HLA class I and II antibody response observed after cryopreserved allograft implantation in children persists for a period of up to 8 years, although it is significantly diminished in this time. There is evidence that the broad HLA specificity (PRA of almost 100%) observed by 3 months after implantation of allograft material may be the result of antibodies directed at high-frequency public epitopes on donor HLA molecules, rather than narrow, private specificities unique to each HLA allele [9, 17, 18]. The persistence of these antibodies likely represents immunologic memory that diminishes with time and in some children becomes undetectable. The disappearance of circulating HLA antibodies may represent a loss of immune memory or, alternatively, it may represent dormant immune memory that could be reactivated with repeat exposure to the offending antigens.

We observed that HLA class II PRA frequency diminished to a greater extent than the HLA class I PRA frequency. Human leukocyte antigen class II PRA was nonreactive in the majority of children (7 of 13) at follow-up compared with HLA class I PRA, which was nonreactive in only 1 of the same 13 children. Although the reason for this difference is unknown, one possible explanation is that dendritic cells of donor origin, known for high levels of both HLA class I and II expression, may have died out relatively early after implantation of the allografts, whereas other donor-derived cells preferentially expressing HLA class I molecules may have remained viable for a longer period. Multiple authors have published reports of viable donor-derived cells found on explanted cryopreserved valved allografts up to several years after implantation [19–21].

The long-term effect of circulating HLA antibodies on allograft function is still speculative. Young children, in particular, have decreased valve survival when compared with adults [22–24], and some have suggested that, among other things, a heightened immune reaction to the implanted tissue may contribute to this. To support this, several studies have described observations consistent with rejection in valves explanted from children, whereas those explanted from adults show little evidence of the same [8, 11]. However, others have observed an almost complete loss of viable donor-derived cells within 2 months of implantation of cryopreserved allografts in children and adults [25]. These authors suggested that circulating HLA antibodies directed at donor-derived cells would therefore not play a significant role in the failure of allografts. Nonetheless, ABO and HLA-DR mismatch and short preservation time (increasing antigenicity of valves) were recently shown to be associated with accelerated allograft valve failure in children [26]. Additional studies are needed to further delineate the role of circulating HLA antibodies in the premature failure of cryopreserved allografts and to determine the benefit, if any, of immunosuppression in the perioperative period on long-term allograft function in children.

The implications of our study are also significant as they relate to the field of heart transplantation. As the number of palliative surgical procedures using cryopreserved allograft material to correct congenital heart malformations increases, a small but significant number of these children will later go on to require heart transplantation. Our study demonstrates a significant level of alloimmune HLA sensitization that persists for many years in children who have received cryopreserved allografts, and leads one to question how this affects long-term survival in children who later require heart transplantation. Several studies have examined the correlation of pretransplant PRA with outcomes after solid organ transplantation in adults. Kidney transplant recipients with elevated PRA have lower graft survival than do patients without such antibodies [27], and several studies have shown a similar effect in adult heart transplant recipients [12–14]. Kobashigawa and colleagues [14] reviewed the pretransplant PRA and outcome after heart transplantation of 311 adult patients who had negative prospective donor-specific crossmatch. Actuarial 3-year survival was 39% for individuals with PRA of more than 10% (n = 20), compared with 3-year survival of 78% for those with PRA of less than 10% (n = 293). Additionally, 35% of those with PRA greater than 10% experienced clinically severe rejection compared with 5% of those with PRA less than or equal to 10%. There is also preliminary data that children with a pretransplant elevation in PRA have increased risk of rejection and decreased survival after heart transplantation [28].

In our study, 12 of 13 children had HLA class I PRA greater than 10% at the time of follow-up. When interpreted in light of the adverse effects of elevated pretransplant PRA, these results suggest that children who undergo a palliative surgical procedure using a cryopreserved allograft may be at increased risk of rejection and mortality should they later require heart transplantation. Hence, new methods of mitigating the antibody response to cryopreserved allograft material warrant investigation. For instance, immune suppression after the implantation of allografts has been successfully used in some children to decrease the HLA response to valved allografts [16], and decellularized allografts have recently been shown to elicit a much diminished PRA reaction compared with cryopreserved allografts [29]. These approaches may lead to increased allograft survival and decreased risk of rejection in children who go on to require heart transplantation.

In summary, recipients of cryopreserved valved and nonvalved allografts mount a broadly reactive donor-specific HLA antibody response that peaks by 3 months and lasts for up to 8 years. This antibody response diminishes with time. It is unclear how the persistence of circulating PRA affects the long-term functionality of cryopreserved allografts. However, owing to a high level of alloimmune presensitization, children who have received cryopreserved allografts may be at higher risk should they later require heart transplantation. Additional studies are warranted to determine the effects of persistent PRA on long-term function of allografts, to develop methods for mitigating the immune response to allograft material, and to determine how previous exposure to cryopreserved allografts affects survival in pediatric heart transplant recipients.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Comment
 References
 

  1. Smith JD, Ogino H, Hunt D, Laylor RM, Rose ML, Yacoub MH. Humoral immune response to human aortic valve homografts Ann Thorac Surg 1995;60(Suppl):S127-30.
  2. Shaddy RE, Hunter DD, Osborn KA, et al. Prospective analysis of HLA immunogenicity of cryopreserved valved allografts used in pediatric heart surgery Circulation 1996;94:1063-1067.[Abstract/Free Full Text]
  3. Hoekstra F, Witvliet M, Knoop C, et al. Donor-specific anti-human leukocyte antigen class I antibodies after implantation of cardiac valve allografts J Heart Lung Transplant 1997;16:570-572.[Medline]
  4. Hoekstra FM, Witvliet M, Knoop CY, et al. Immunogenic human leukocyte antigen class II antigens on human cardiac valves induce specific alloantibodies Ann Thorac Surg 1998;66:2022-2026.[Abstract/Free Full Text]
  5. Hawkins JA, Breinholt JP, Lambert LM, et al. Class I and class II anti-HLA antibodies after implantation of cryopreserved allograft material in pediatric patients J Thorac Cardiovasc Surg 2000;119:324-330.[Abstract/Free Full Text]
  6. Breinholt III JP, Hawkins JA, Lambert LM, Fuller TC, Profaizer T, Shaddy RE. A prospective analysis of the immunogenicity of cryopreserved nonvalved allografts used in pediatric heart surgery Circulation 2000;102(19 Suppl 3):III-179-82.
  7. Rajani B, Mee RB, Ratliff NB. Evidence for rejection of homograft cardiac valves in infants J Thorac Cardiovasc Surg 1998;115:111-117.[Abstract/Free Full Text]
  8. Vogt PR, Stallmach T, Niederhauser U, et al. Explanted cryopreserved allografts: a morphological and immunohistochemical comparison between arterial allografts and allograft heart valves from infants and adults Eur J Cardiothorac Surg 1999;15:639-645.[Abstract/Free Full Text]
  9. Shaddy RE, Thompson DD, Osborne KA, Hawkins JA, Fuller TC. Persistence of human leukocyte antigen (HLA) antibodies after one year in children receiving cryopreserved valved allografts Am J Cardiol 1997;80:358-359.[Medline]
  10. Dignan R, O'Brien M, Hogan P, et al. Aortic valve allograft structural deterioration is associated with a subset of antibodies to human leukocyte antigens J Heart Valve Dis 2003;12:382-391.[Medline]
  11. Rajani B, Mee RB, Ratliff NB. Evidence for rejection of homograft cardiac valves in infants J Thorac Cardiovasc Surg 1998;115:111-117.
  12. Lavee J, Kormos RL, Duquesnoy RJ, et al. Influence of panel-reactive antibody and lymphocytotoxic crossmatch on survival after heart transplantation J Heart Lung Transplant 1991;10:921-930.[Medline]
  13. Loh E, Bergin JD, Couper GS, Mudge Jr GH. Role of panel-reactive antibody cross-reactivity in predicting survival after orthotopic heart transplantation J Heart Lung Transplant 1994;13:194-201.[Medline]
  14. Kobashigawa JA, Sabad A, Drinkwater D, et al. Pretransplant panel reactive-antibody screensAre they truly a marker for poor outcome after cardiac transplantation?. Circulation 1996;94(9 Suppl 2):II-294-7.
  15. Shaddy RE, Lambert LM, Fuller TC, et al. Prospective randomized trial of azathioprine in cryopreserved valved allografts in children Ann Thorac Surg 2001;71:43-48.[Abstract/Free Full Text]
  16. Shaddy RE, Fuller TC, Anderson JB, et al. Mycophenolic mofetil reduces the HLA antibody response of children to valved allograft implantation Ann Thorac Surg 2004;77:1734-1739.[Abstract/Free Full Text]
  17. Fuller TC. Monitoring HLA alloimmunizationAnalysis of HLA alloantibodies in the serum of prospective transplant recipients. Clin Lab Med 1991;11:551-570.[Medline]
  18. Oldfather JW, Gebel HM, Holmes DJ, Rodey GE. Detection of two distinct public specificities on HLA-A28 bearing lymphocytes Tissue Antigens 1983;22:16-23.[Medline]
  19. Lupinetti FM, Tsai TT, Kneebone JM, Bove EL. Effect of cryopreservation on the presence of endothelial cells on human valve allografts J Thorac Cardiovasc Surg 1993;106:912-917.[Abstract]
  20. Armiger LC. Postimplantation leaflet cellularity of valve allografts: are donor cells beneficial or detrimental? Ann Thorac Surg 1998;66(6 Suppl):S233-5.
  21. O'Brien MF, Stafford EG, Gardner MA, Pohlner PG, McGiffin DC. A comparison of aortic valve replacement with viable cryopreserved and fresh allograft valves, with a note on chromosomal studies J Thorac Cardiovasc Surg 1987;94:812-823.[Abstract]
  22. Hawkins JA, Bailey WW, Dillon T, Schwartz DC. Midterm results with cryopreserved allograft valved conduits from the right ventricle to the pulmonary arteries J Thorac Cardiovasc Surg 1992;104:910-916.[Abstract]
  23. Clarke DR, Campbell DN, Hayward AR, Bishop DA. Degeneration of aortic valve allografts in young recipients J Thorac Cardiovasc Surg 1993;105:934-942.[Abstract]
  24. Tweddell JS, Pelech AN, Frommelt PC, et al. Factors affecting longevity of homograft valves used in right ventricular outflow tract reconstruction for congenital heart disease Circulation 2000;102(19 Suppl 3):III-130-5.
  25. Mitchell RN, Jonas RA, Schoen FJ. Structure-function correlations in cryopreserved allograft cardiac valves Ann Thorac Surg 1995;60(2 Suppl):S108-13.
  26. Baskett RJ, Nanton MA, Warren AE, Ross DB. Human leukocyte antigen-DR and ABO mismatch are associated with accelerated homograft valve failure in children: implications for therapeutic interventions J Thorac Cardiovasc Surg 2003;126:232-239.[Abstract/Free Full Text]
  27. Terasaki PI, Cecka JM, Takemoto S, et al. Clinical transplants 1988. Overview. Clin Transpl 1988:409–34..
  28. Pediatric Heart Transplant Study GroupMahle WT, Naftel DC, Rusconi P, Edens RE, Shaddy RE. Panel-reactive antibody cross-reactivity and outcomes in the Pediatric Heart Transplant Study Group J Heart Lung Transplant 2004;23(Suppl 2):S167.
  29. Hawkins JA, Hillman ND, Lambert LM, et al. Immunogenicity of decellularized cryopreserved allografts in pediatric cardiac surgery: comparison with standard cryopreserved allografts J Thorac Cardiovasc Surg 2003;126:247-253.[Abstract/Free Full Text]



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