HLA-Expression-in Aortic and Pulmonary 
Homografts: Effects of Cryopreservation

Susan Verghese, KM Cherian

Institute of Cardiovascular Diseases, Madras Medical Mission, Chennai

Background: The present study was undertaken to find out the HLA allo-antigens on cardiac homografts. Methods and Results: One pulmonary and eight aortic homografts were studied for the presence of major HLA class I and class II antigen expression. Cadaveric hearts were procured from the mortuary and kept in Hank’s balanced salt solution with antibiotics at 4°C. Bits were taken from the conduits and valves every 24 hours for 14 days during storage and snap-frozen using liquid nitrogen. A total of 1368 sections were made using a cryostat. These sections were stained using 4 monoclonal antibodies: HLA class I (M0736), class II HLA-DR (M0746), CD45 (M0701), and endothelial stain (M0616). All monoclonals were procured from DAKO. Class I antigen molecules could be demonstrated on the endothelial surface of the vessel wall from day 1 to day 4 to 5 of storage. They stained weaker and could not be demonstrated after day 10 of storage. Class I antigen molecules were positive in very fresh valves and by day 5–6 could not be seen on the valve surface. Class II (HLA-DR) antigen expression was present in the subendothelial layer from day 1 to day 12–14 of storage. They could also be demonstrated in valves and conduits released after cryopreservation. These class II staining cells were also stained by CD45 monoclonal antibody and hence could be macrophages, histiocytes or leucocytes.  The endothelium was very well demonstrated in the vessel walls from day 1 to day 12–14 of storage; it could only be seen in very fresh valves. Storage in the liquid medium and sterilization procedures led to loss of endothelial lining of the valves. After cryopreservation and thawing, class I antigen molecules could not be demonstrated on the valves and conduits. Class II antigen molecules and CD45-stained cells continued to be demonstrated in the subendothelial layer and the valve matrix. The endothelium was intact in the vessel wall after cryopreservation and thawing, but could not be seen in the released valves.

Conclusions: Allograft aortic and pulmonary conduits and valves are immunogenic, and HLA-ABC and HLA-DR antigen molecules can be demonstrated on different components of the vessel wall and valve leafets. (IndianHeart J 2002; 54: 394–398)

Key Words: HLA antigens, Cardiac homografts, Cryopreservation 

Allograft valves and conduits used for replacement of diseased and damaged valves in adults and children have shown clinical durability except in children.1 However, there is concern regarding the long-term performance of the valves. The role of immunological damage in producing degenerative valve failure has not been adequately studied.There have been studies in which homografts have been shown to express both class I and class II antigens.2 This expression is known to decrease with the time of immersion in the antibiotic fluid. It has been suggested that the durability of allograft valves depends on fibroblast viability of the valves at implantation.1 Currently, fibroblast viability appears to be best achieved by the gentle harvesting method, careful cryopreservation using DMSO and processing valves at the earliest.3

Homografts have been used in very few centers in India and most of these are procured from the mortuary from victims of sudden death at the time of autopsy. India being a tropical country, the rate of degeneration of the valves prior to collection is probably high. The antibiotics used and media used for cryopreservation are also different from those used in western countries. Hence this study was undertaken to demonstrate the HLA allo-antigens on cardiac homografts and to demonstrate the effect of storage and cryopreservation on them.

Methods

One pulmonary and eight aortic valves and conduits were examined. The cadaveric hearts were collected from the mortuary within 24 hours after death and transported in Hank’s balanced salt solution (HBSS) with antibiotics. The age group of the donors ranged from 27 to 45 years. There were 7 males and 2 females. All the valves and conduits appeared normal. Two bits were taken from the valve and the conduits everyday, starting from 24 hours after death.The specimens were placed in a tissue paper with a drop of tissue-embedding solution (Jung tissue freezing medium) and snap-frozen by immersion in liquid nitrogen. They were then stored in the vapour phase of liquid nitrogen until sections were made using a cryostat (Leica CM 1100). The procedure was repeated at intervals of 24 hours till the end of the day 14. During this period, the homograft was stored in HBSS with antibiotics at 4 °C. The antibiotics/antifungals used in the medium were the same as those used to sterilize homografts in our Homograft-Valve-Bank.

Ten bits of previously cryopreserved tricuspid valve frozen in MI99 with 10% DMSO, upon thawing, were washed in MI99 with 5% DMSO and then twice with plain MI99. These bits were snap-frozen in a manner similar to that described above and sections of the tricuspid valve bits were also taken using a cryostat.

Five aortic conduits and aortic valves which had been frozen in MI99 with 10% DMSO were thawed and rinsed in a similar manner, and bits and sections were taken from the aortic walls and valve bits.

All the sections were air-dried for 24 hours at room temperature, wrapped in silver foil and stored at –20 °C till they were stained.

 The sections were fixed in acetone for 10 min prior to staining. Antigen expression was evaluated by monoclonal antibodies using the alkaline phosphatase–anti-alkaline phosphatase (APAAP) method. The procedure used was the same as described by Yacoub et al.4 Briefly, the sections were incubated with mouse monoclonal antibody (MAb). They were then washed with Tris buffered saline (TBS) and incubated with anti-mouse antibody, washed again with TBS followed by incubation with APAAP complex. After a further incubation and wash, the substrate was finally added (Fast Red). The substrate was incubated on the section for 20 min and washed with TBS and tap water.The slide was finally mounted using aqueous mounting fluid. The stained sections were studied immediately. The staining was graded as (+) (weak) to (++++) (very strong), depending on the intensity of the stain and recorded.

  The monoclonal antibodies used in the study were; (i) Mouse monoclonal anti-human HLA class I antigen (M0736) at a concentration of 1/50 in TBS; (ii) Mouse monoclonal anti-human class II HLA-DR antigen (M0746) at a concentration of 1/50 in TBS; (iii) Mouse monoclonal anti-leucocyte common antigen (CD45) at a concentration of 1/50 in TBS; (iv) Mouse monoclonal anti human vonWillebrand factor (M0616) at a concentration of 1/50 in TBS, which was used to stain the endothelium. 

Results 

The total number of fresh conduits and valves studied was 9. The number of sections of conduits and valves that were sectioned and stained from each homograft was 152 (4×14 for conduit and 4×14 for valve). The total number of sections stained from fresh valves was 1368. 

HLA class I antigen expression: Aortic conduits from autopsy material showed positive staining of the endothelial surface ranging from very strong (++++) (Fig. 1) to strong (++) from day 1 to day 4–5 in HBSS with antibiotic (AB). The class I antigen expression becomes weaker from day 4 to 5 but could be demonstrated up to day 8–9 (+) in most vessel walls.The valves showed strong staining on day 1–2 on the endothelial surface (+++). Class I antigen molecules could not be demonstrated after day 5–6 in all valves. Class I antigen expression could also be demonstrated in the microvasculature of the adventitia of the aortic vessel walls. Class I antigen molecules could not be demonstrated in the vessel walls or adventitia or valve surface after day 10 (Fig. 2).

Class II antigen expression: Cells stained by HLA-DR were present of the subendothelial layer of the aortic wall from day 1 to day 12–14. The staining was very strong (++++) (Fig. 3) to strong (++) by day 12–14 (Fig. 4). Class II antigen-stained cells could also be found in the valve matrix (in the center of the valve cusp) (Fig. 5) and just below the endothelium of the valve lining (Fig. 6). These valve matrix cells could be seen staining up to day 8–10 with HLA-DR MAb.

CD45 antigen expression: Cells stained by CD45 MAb, in the subendothelial layer of the vessel wall could be demonstrated up to day 12–14 of storage. The cells in the subendothelial lining which had stained by the class II antigen molecules also stained by CD45 antigen (common leucocyte antigen) (Fig. 7) as did the cells in the valve matrix. The HLA-DR stained cells were probably leucocytes, macrophages or histiocytes as the CD45 MAb stains these cells. CD45 MAb did not stain the endothelial surface of the vessel walls or valves which had HLA-ABC class I antigen expression.

Endothelial stain: The aortic wall lining of the endothelium was intact up to day 14 (Figs 8 and 9) of storage in HBSS with AB, and could be well stained by the endothelial stain. Thus class I antigen expression was independent of the endothelial lining.

Valves: Endothelium was present in fresh valves. But with continued storage in HBSS with AB the endothelium could not be seen after day 4–5 of storage.

After cryopreservation and thawing: Class I antigen expression could not be demonstrated on the vessel walls or on the valves after cryopreservation. Class II antigen expression could be demonstrated in the subendothelial layer of the aorta, which had been cryopreserved and thawed. Class II antigen-stained cells could also be seen to be weakly staining (+) in the valve matrix after cryopreservation and thawing. These cells could also be stained using CD45 MAb. CD45-stained cells could be seen in the subendothelial layer and in the valve matrix. These were similar to the cells stained by class II antigen molecules. The endothelium in the vessel walls stained positive even after cryopreservation and thawing. In the valves, the endothelium could not be demonstrated after thawing. The procedure of sterilization and freezing and thawing probably destroyed the thin one-celled layer of the endothelium on the valves.

Discussion

The early series of homografts inserted between 1962 and 1969 were sterilized by beta-propiolactone, ethylene oxide and gamma radiation. Being dissatisfied with the medium-term clinical results after using chemically sterilized homografts, Barrett-Boyes 5 introduced antibiotic sterilization with high-dose antibiotics. Subsequently, the dosage was reduced to decrease cytotoxicity. To achieve a higher viability rate of heart valve allografts during long-term preservation in nutrient medium, cryopreservation techniques were adopted. Refinement of cryopreservation techniques maintains cellular and tissue viability and eliminates the possibility of structural damage as a factor for allograft degeneration. On the other hand, it creates an immunological problem. With cryopreserved heart valves, a higher rate of viable endothelial cells and the presence of surface antigens were observed which could cause an immunological reaction. The significance of endothelial cells, fibroblasts and the immunological basis of degeneration has not been studied extensively. This study was undertaken to demonstrate the expression of HLA class I and class II antigens on homograft conduits and valves, and to study the effect of sterilization procedures, storage and cryopreservation on them. 

We were able to demonstrate class I antigen molecules on the endothelial surface of the vessel walls and also on the endothelial lining of the vessels in the adventitia. The staining was intense in fresh conduits and valves. Class I antigen expression became less intense after day 4–5 and could not be demonstrated after day 10 of storage. Class I antigen expression was independent of the presence of endothelial lining. Yacoub et al.4 demonstrated the presence of major HLA class I and class II antigen molecules on the valve surface and on the aortic wall endothelial surface.But they were unable to demonstrate the antigen expression after 48 hours of storage in the nutrient medium. Antigenicity of allograft conduits and valves has been demonstrated in other studies.6 Besides expression of HLA class I and class II antigen molecules, dendritic cells have also been demonstrated in a few studies.7 Class II antigen-expressing cells were found to be situated in the sub-endothelial layer in the aortic vessel wall and in the valve matrix. We have been able to show the presence of these cells up to day 14 of storage in the vessel walls and also in the valve matrix. These cells could be responsible for mounting a detrimental immune response. They were also stained by common leucocyte antigen (CD45). Hence, these class II antigen-bearing cells could be leucocytes, macrophages and histiocytes. The presence of dendritic cells has not been confirmed in the literature but Bobryshev et al.8 have reported the presence of dendritic cells in the aortic vessel walls. The vessel wall represents an important component of the valve allograft.

The fact that homografts are antigenic has also been demonstrated by a few authors. Smith et al.9 have shown that recipients of homovital aortic valve homografts are known to produce donor-specific antibodies to the cellular components of valve tissue. These antibodies have been found to persist for long periods of up to 15 years and they have been found to be of the IgG class. Further studies may be needed to determine the relationship between antibody status and graft longevity and also to determine if immunosuppressive medication or preoperative tissue typing and matching can alter the immune response. 

Endothelial cells have not been demonstrated consistently on cryopreserved valve leaflets. Lupinetti et al.10 showed that cryopreservation of human donor valves is associated with loss of endothelial cells. This has also been the experience of Hoekstra et al.11 and Mitchell et al.12 where only a few endothelial cells could be demonstrated on the valve leaflets and vascular structures. Our study differs from theirs in that the endothelium could be demonstrated on the aortic vessel wall up to day 12–14 of storage in HBSS with AB. But we were not able to demonstrate the same in the valve leaflet. The endothelium could only be seen in very fresh valves. After cryopreservation and thawing, endothelium could be demonstrated in the aortic vessel wall but not on the valve leaflet.

Allograft preparation methods used in different centers differ in the antibiotics and nutrient medium used, varying ischemic time before the valves are collected and the duration of AB sterilization. This could lead to different antigenic load on implanted valves.

Conclusions: The durability of homograft valves may be graft- or patient-related. One of the important variables is the antigenicity of different components of the graft at the time of insertion. Allograft aortic and pulmonary conduits and valves are immunogenic. We have demonstrated HLA-ABC (class I) and HLA-DR (class II) antigen molecules on different components of the vessel wall and valve leaflets. 

Acknowledgments

The authors are deeply grateful to Rameshwardas Birla Smarak Kosh, Mumbai for their financial assistance. We acknowledge the technical help of microbiologists P Padmaja, T Mathew and CF Maria. 

Correspondence: 
Dr KM Cherian, Director, 
Institute of Cardiovascular Diseases, 
Madras Medical Mission, 
4-A, JJ Nagar, Mogappair, 
Chennai

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