Ann Thorac Surg 2009;87:211-214. doi:10.1016/j.athoracsur.2008.09.073
© 2009 The Society of Thoracic Surgeons
Original Articles: Pediatric Cardiac
Expression of A and B Blood Group Antigens on Cryopreserved Homografts
Brian Feingold, MDa,*,
Peter D. Wearden, MD, PhDb,
Victor O. Morell, MDb,
Daniel Galvis, BS, PA (ASCP)c,
Csaba Galambos, MDc
a Department of Pediatric Cardiology, Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania
b Department of Cardiothoracic Surgery, Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania
c Department of Pathology, Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania
Accepted for publication September 29, 2008.
* Address correspondence to Dr Feingold, Division of Pediatric Cardiology, Children's Hospital of Pittsburgh of UPMC, 3705 Fifth Ave, Pittsburgh, PA 15213 (Email: brian.feingold{at}chp.edu).
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Abstract
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Background: The contribution of ABO blood group mismatching on the rate of cardiac homograft failure is uncertain. It has been shown that there is lack of expression of A and B blood group antigens on fresh and cryopreserved homograft valves. However, expression of these antigens on the homograft vessel conduit has not been reported.
Methods: Unused portions of cryopreserved pulmonary artery homografts (n = 16) were immunohistochemically stained and examined for expression of endothelial cell marker CD31 and A and B blood group antigens. The staining pattern for each antigen was described. Comparison of homograft donor blood group (as determined by immunostaining) to the blood group reported by the homograft supplier was made.
Results: Staining of CD31 and A and B blood group antigens was poor on the luminal surface (tunica intima) of the homograft conduit but strong in the vaso vasorum of the tunica media and adventitia. Homograft donor blood group was consistent with the reported donor blood group in 15 of 16 specimens (4 group A, 3 group B, 8 group O). In one specimen (reported as group O), we detected strong expression of CD31 and A antigen on the endothelium of the vaso vasorum.
Conclusions: Cryopreserved homografts strongly express A and B blood group antigens on well-preserved endothelial surfaces of the medial and adventitial vaso vasorum. The significance of this finding with regard to the immunologic response to mismatched ABO blood group antigens and homograft longevity is uncertain.
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Introduction
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There are conflicting data on the contribution of ABO blood group mismatching to the rate of homograft failure. Whereas some authors have concluded placement of an ABO incompatible homograft has no impact on failure rates [1, 2], others have found blood group incompatibility between homograft donor and recipient a significant risk factor for accelerated homograft failure [3–5]. In a study of blood group antigen expression on cardiac valve endothelium, Kadner and colleagues [6] found no ABH expression on either fresh or cryopreserved homograft valves and thus concluded that blood group incompatibility was unlikely to play a significant role in homograft degeneration. However, typically more than just the valve is utilized in the repair of congenital cardiac lesions (Ross procedure, aortic arch reconstruction for first stage of hypoplastic left heart syndrome palliation, truncus arteriosus, etc). If mismatched blood group antigens are present in the conduit, an immune response to these antigens might result in alterations in conduit geometry resulting in stenosis or insufficiency, which could impact homograft outcome. Because there are no reported data on the expression of A and B blood group antigens within the homograft conduit, we undertook this study.
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Material and Methods
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This study was approved by the University of Pittsburgh Committee for Oversight of Research Involving the Dead. Unused portions of 16 consecutively implanted, cryopreserved vascular homografts (LifeNet Health, Virginia Beach, VA) were collected from the operating table and immediately placed into RPMI-1640 medium (Invitrogen, Carlsbad, CA). Specimens were refrigerated in RPMI at 4°C for up to 24 hours before processing. All homografts were selected for use based on clinical-surgical indications and only the trimmed portions (to be otherwise discarded) were collected for study. Prior to selection for clinical use, each homograft was stored and thawed according to the distributor's guidelines. Donor (homograft) blood group was ascertained from the clinical documentation provided with the homograft.
Homografts were sampled for routine tissue processing including formalin fixation and paraffin-embedding. Immunoperoxidase staining with primary anti-A and B (murine monoclonal, series 1) blood group antibodies (Immucor, Norcross, GA) was performed using the Ventana Benchmark XT automatic slide stainer (Ventana Medical Systems, Tucson, AZ) with no antigen retrieval. Similarly, preservation of endothelium was separately confirmed by staining for CD31 using murine monoclonal (clone JC70A) antibody (Dako, Carpinteria, CA). Slides were incubated with primary antibody for 32 minutes at 37°C, then with the I-VIEW DAB detection system (Ventana), and counterstained with hematoxylin. Antibodies were diluted in Tris- bovine serum albumin-buffered solutions with the following dilutions: anti-A at 1:400, anti-B at 1:400, and CD31 at 1:200. For each antibody, positive results required diffuse granular membranous brown staining. Complete absence of endothelial staining was the requirement for negative cases. Positive and negative controls were run in each batch and deemed adequate. All specimens were reviewed by a single pathologist who was blinded to the donor blood type.
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Results
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All homografts (n = 16) were cryopreserved pulmonary artery. Median donor age was 35 years (range, 2 months to 50 years). Donor gender was 4 females and 12 males. Four homografts were from blood group A donors, 3 from blood group B donors, and 9 from blood group O donors.
Table 1
shows the results of staining by anti-A, anti-B, and anti-CD31 antibodies for both the luminal surface and subluminal portions (tunica media and tunica adventitia) of the homograft conduits. In general, the luminal surface showed only patchy or minimal expression of CD31 and blood group antigens (Table 1 and Figs 1A,
1C, 1E), whereas the vascular subluminal region demonstrated diffuse staining for CD31 and blood group antigens (Figs 1B, 1D, 1F). In all specimens there was concordance between luminal and subluminal staining. Donor blood group was accurately predicted by immunoperoxidase staining in 15 of 16 cases. In the one case with a discrepancy, immunoperoxidase staining predicted the homograft donor blood group as type A whereas the homograft donor was reported as blood group O by the distributor.
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Fig 1. Immunohistochemical staining with CD31 (A, B), anti-A (C, D), and anti-B (E, F) antibodies are shown. Endothelial cell staining of homograft lumen is minimal and focal with all antibodies (A, C, E; arrows). In contrast, endothelial cells of small vessels located in tunica media and adventitia stain strongly and diffusely with CD31 as well as anti-A and anti- B antibodies (B, D, F; arrows). Hematoxylin counterstain, magnification x40 (A, C, E) and x10 (B, D, F).
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Comment
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This report is the first to describe A and B blood group antigen expression on homograft vessel conduits. In the only other report detailing blood group antigen expression on cryopreserved homografts, Kadner and colleagues [6] showed that ABH antigens and CD31 were not detectable on cryopreserved homograft valves but no examination of the homograft conduit was performed. ABH antigens were also not found on fresh cardiac valve endothelium despite uniformly positive CD31 staining. These data suggest that ABH expression does not occur on valve endothelium, in contrast to other vascular endothelial surfaces. In the current study we have shown that although the luminal surface of cryopreserved homograft conduits poorly express both CD31 and the A and B antigens (likely due to poor endothelial survival), there is strong and consistent expression of both CD31 and A and B antigens (as appropriate for donor blood group) in the tunica media and adventitia vaso vasorum of the conduit.
The significance of donor and recipient ABO mismatching as a risk factor for accelerated homograft failure is uncertain. In an early study, Shaddy and colleagues [7] found blood group incompatibility between homograft donor and recipient was not associated with the development of calcification or stenosis in 39 children who received valved allografts. More recently Christensen and coworkers [4] concluded that ABO mismatching was associated with the development of accelerated fibrocalcifications. There are numerous other conflicting reports in the literature associating [3, 5] or not finding an association of [1, 2] ABO mismatching with progression to homograft failure. In each of these studies, endpoints such as progression of stenosis-insufficiency or homograft replacement were assessed by echocardiography or time to reoperation. The difficulty in interpreting these retrospective studies is the lack of consistency for defining homograft failure by imaging and clinical criteria. Using a different approach, Fischlein and colleagues [8] showed greater in vivo cytoimmunologic activity in patients who received ABO mismatched versus matched homografts.
In infants and children, homografts are commonly used as conduits or patches rather than solely as replacement valves. Thus, one could theorize that an immunologic response directed against mismatched ABO antigens expressed on the homograft could effect conformational changes and result in stenosis and(or) insufficiency. It is of interest that expression of both class I and class II HLA antigens in cryopreserved homografts has been shown to also be distributed in the tunica media and adventitia, with only patchy expression on the luminal surface (class I) and valve matrix (class II) [9, 10]. It is also well accepted that after cryopreservation homografts are a source of immunostimulatory cells [11–13] and a potent cause of anti-human leukocyte antigen allosensitization [14–16]. Whether or not the stimulus from similarly distributed A and B blood group antigens is sufficient to contribute to immune mediated injury to the homograft or to impair development of anti-A and(or) anti-B antibodies, as seen in infant ABO incompatible heart transplantation [17, 18], is uncertain.
The single discrepancy in homograft blood group classification we observed may be explained by a clerical error in blood type reporting by the distributor. Alternately, it is possible that the donor was mistyped due to the presence of a nondeletional O allele resulting in "weak" A expression [19–21].
In summary, cryopreserved human pulmonary artery homografts strongly express A and B blood group antigens on well-preserved endothelial surfaces of the medial and adventitial vaso vasorum. We found only scant A and B expression on homograft conduit luminal surfaces with limited endothelial cell (CD31) staining. Although the significance of these findings in terms of the immunologic response and impact on homograft longevity are unclear, our results do provide scientific plausibility to the theory of immune mediated injury directed against mismatched A and B blood group antigens leading to more rapid homograft failure.
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Acknowledgments
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The authors would like to thank Kent Kelly, CCP, and Cindy Cardwell, RN, for their aid in securing homograft specimens, Connie Riga for her assistance in performing immunohistochemical staining, and Darrell J. Triulzi, MD, and James Cramer for providing anti-A and B antibodies.
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References
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M. L. Jacobs
Invited Commentary
Ann. Thorac. Surg.,
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87(1):
214 - 215.
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Abstract
Introduction
