Ann Thorac Surg 2001;71:S349-S352
© 2001 The Society of Thoracic Surgeons
Autografts, allografts, and biological valves in children
Homograft crossmatching is unnecessary due to the absence of blood group antigens
Alexander Kadner, MDa,
Raymond H. Chen, MD, PhDa,
Richard N. Mitchell, MD, PhDb,
David H. Adams, MDa
a Division of Cardiac Surgery, Brigham & Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA
b Department of Pathology, Brigham & Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA
Address reprint requests to Dr Adams, Division of Cardiac Surgery, Brigham & Women’s Hospital, 75 Francis St, Boston, MA
e-mail: dadams{at}partners.org
Presented at the VIII International Symposium on Cardiac Bioprostheses, Cancun, Mexico, Nov 3–5, 2000.
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Abstract
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Background. Homograft valves are subject to calcification and structural degeneration in the long term. Blood group matching is performed in many centers, and it remains controversial whether immunologic responses associated with potential blood group incompatibility contribute to the degeneration of unmatched homografts. We studied the expression of carbohydrate blood group antigens on valve endothelium of thawed aortic homograft valves and freshly harvested human cardiac valves.
Methods. Cryopreserved human aortic homograft valves and freshly harvested human aortic, pulmonary, mitral, and tricuspid valves were incubated with antibodies to A, B, and O blood group antigens.
Results. Cardiac microvascular endothelium stained positively with antiendothelial CD31 antibody in both cryopreserved and fresh tissue. Cryopreserved valve endothelial lining rarely stained positively for CD31, in contrast to fresh valves, which always stained positive. Cryopreserved or fresh cardiac microvascular endothelium strongly expressed A, B, or H antigens. In contrast, ABH antigens were not detectable on homograft or fresh cardiac valve endothelium.
Conclusions. The absence of expression of carbohydrate antigen on valvular endothelium suggests that blood group incompatibility does not play a significant role in homograft degeneration.
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Introduction
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Homograft valves are increasingly used for aortic valve replacement. They present several advantages compared with mechanical valves such as an exceptionally good hemodynamic performance, lack of postoperative anticoagulation, freedom from thrombosis and thromboembolic events, and a relative resistance to endocarditis [1]. However, homograft valves are susceptible to calcification and structural degeneration in the long term [2]. The pathophysiologic mechanism of homograft failure is not well understood and it has been postulated that immunologic phenomena may contribute to the degeneration process. Experiences from allotransplantation have shown that recipient–donor blood group incompatibility elicits a strong rejection response, and, consequently, blood group crossmatching is performed in many centers. However, it remains controversial whether immunologic responses associated with potential blood group incompatibility contribute to the degeneration of unmatched homograft valves [3].
To clarify this issue, we characterized the expression of blood group antigens on valve endothelium of cryopreserved aortic homograft valves and extended our study of freshly harvested heart valves from transplant recipients [4].
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Material and methods
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Cryopreserved human homografts
Cryopreserved human aortic homograft valves from blood group A, B, and O (n = 1 each blood group) were obtained from American Red Cross Inc (Irvine, CA). The homograft valves were stored in liquid nitrogen and thawed according to the instruction of the manufacturer. Sections of aortic valve and myocardium were analyzed.
Fresh human heart valves
Human aortic, pulmonary, mitral, and tricuspid valves and myocardial tissue were freshly harvested from explanted hearts of patients undergoing heart transplantation or valve replacement. Thirteen aortic valves (blood group A, 6; B, 3; O, 4), eight mitral valves (blood group A, 4; B, 1; O, 3), six tricuspid valves (blood group A,3; O, 3), and six pulmonary valves (blood group A, 3; O, 3) were available for analysis.
Tissue fixation and preparation
Valvular and myocardial sections were fixed in a Carnoy’s solution or in phosphate-buffered formalin and embedded in paraffin. Paraffin sections (5 µm thick) were washed four times for 4 minutes in Histoclear baths to remove paraffin embedding, followed by washing two times for 2 minutes in 100% and subsequently 95% ethanol. After incubating with distilled water for 5 minutes, tissue samples were treated with 3% H2O2 and washed for 5 minutes in phosphate-buffered saline buffer.
Immunohistochemistry
Tissue sections were incubated with monoclonal mouse antibodies specific for anti-A (clone 81FR 2.2), anti-B (clone 3E7), and anti-H (clone 92FR-A2) blood group antigens (DAKO, Carpinteria, CA) for 1 hour followed by washing in phosphate-buffered saline buffer for 5 minutes. Incubation with a biotin-labeled goat antimouse IgG antibody (Sigma Chemical Co, St. Louis, MO) was performed for 1 hour followed by washing with phosphate-buffered saline buffer for 5 minutes. The signal was developed with the avidin-peroxidase system (ABC kit, Vector Lab, Burlingame, CA). The preservation of endothelium was separately confirmed by staining for CD31 (monoclonal mouse anti-CD31, clone JC/70A, Sigma Chemical Co) on formalin-fixed valves and myocardium.
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Results
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Cryopreserved human aortic homograft valves and myocardial tissue
Blood group A, B, or H antigens were not detected on thawed aortic homografts (Fig 1A). Septal myocardial tissue obtained from the same cryopreserved homografts demonstrated uniform expression of A, B, or H carbohydrate antigens on microvascular endothelium (Fig 1B). Thawed homograft leaflets also failed to stain for CD31 (Fig 1C).
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Fig 1. Cryopreserved aortic homograft valves and myocardial tissue. Whereas blood group B antigens were not detected on thawed aortic homograft leaflets (A), B antigens were found on thawed myocardial microvasculature (B). The loss of valve endothelial layer was confirmed by the absence of CD31 staining (C). (Hematoxylin counterstain, magnification x100.)
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Fresh human heart valves and myocardial tissue
Freshly explanted myocardial tissue strongly expressed A (Fig 2A), B (Fig 2B), and H (Fig 2C) antigens. In contrast, blood group antigens A, B, and H could not be detected on pulmonary (Fig 2D), mitral (Fig 2E), and aortic (Fig 2F) valvular endothelium. The preservation of valvular endothelium was demonstrated by strong positive staining of CD31 on pulmonary (Fig 2G), mitral (Fig 2H), and aortic (Fig 2I) valves.
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Fig 2. Fresh human heart valves and myocardial tissue. Blood group antigens A (A), B (B), and H (C) were readily detected on myocardial microvasculature. Blood group antigens, however, were not detected on valve leaflets. Blood group antigens A, B, and H were not found on pulmonary (D), mitral (E), and aortic (F) valve leaflets, respectively. Valvular endothelial preservation on pulmonary (G), mitral (H), and aortic (I) valve leaflets were confirmed by CD31 staining. (Hematoxylin counterstain, magnification x100.)
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Comment
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Homograft valves are subject to calcification and structural degeneration in the long term. It has been suggested that homograft valve failure may result from immunologic rejection [5]. Consequently, blood group matching is performed in many centers. However recipient–donor crossmatching before homograft implantation remains controversial in several studies [6].
The statistical significance of blood group incompatibility as a predictor of valve failure was shown in a study by Yankah and colleagues [7], who found in a group of 42 children that ABO incompatibility was an independent risk factor for early homograft degeneration. By recording the immune response by cytoimmunologic monitoring of peripheral blood in 33 patients, Fischelin and associates [8] demonstrated a more intense response after insertion of ABO-incompatible homografts in 16 patients.
In contrast, Balch and Karp [4] could not show any relationship between ABO blood group compatibility and homograft failure in a group of 30 patients (11 mismatched homografts). Similarly, Yacoub [9] reported no difference between patients with or without ABO incompatibility.
To clarify this controversy, we studied the expression of blood group antigens on human valve endothelium of thawed cryopreserved homograft valves and freshly harvested heart valves from transplant recipients. Cryopreserved homograft valves no longer possess an endothelial layer, as seen by the loss of CD31 signals. These findings are consistent with the report by Mitchell and colleagues [10] in a study of 14 thawed homograft valves.
In contrast, the endothelial layer of freshly harvested valve leaflets was well preserved, confirmed by positive CD31 staining. Immunohistochemistry showed a strong expression of ABH antigens on myocardial microvascular endothelium. However, no ABH antigens could be detected on valve endothelium; thus, it appears that blood group antigens are differentially expressed on cardiac microvascular and valvular endothelium.
In summary, we conclude that the absence of blood group antigen expression on valve endothelium suggests that blood group incompatibility is unlikely to play a significant role in homograft degeneration. Avoidance of blood group crossmatching could increase the pool of available homograft valves for patients with less frequent blood groups.
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Acknowledgments
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The authors thank Jay Tracy for his excellent technical assistance. Doctor Chen is an American College of Surgeons Research Scholar 1998–2000 and recipient of NIH Individual National Research Service Award (NRSA) 1F32HL0996601.
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References
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Yacoub M.H. Applications and limitations of histocompatibility in clinical cardiac valve allograft surgery. In: Yankah A.C., Hetzer R., Miller D.C., Ross D.N., Somerville J., Yacoub M.H., eds. Cardiac valve allografts 1962–1987. New York, NY: Springer Verlag, 1987:89-94.
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Abstract
Introduction
