The Effect Of Cryopreservation Late-pregnancy Fetus Homograft Valved Conduit Immunogenicity

Objective: The present study was undertaken to analysis the HLA alloantigen expression on cardiac homografts.To evaluate the effect of cryopreservation on immunogenicity of the homografts aortic and pulmonary conduits and valves from late-pregnancy fetus.Methods: Aortic and pulmonary HVCs were procured from 3 male and 7 female brain death fetus (with gestational age more than 28 weeks,WIT within 12 hours).Every specimen cut into two portions,the fresh one portion was control group and were quickly frozen with embedding agent ZLI-9302,another was experiment group and was thawed after 2 months cryopreservation with M199 and DMSO and then quickly frozen with embedding agent ZLI-9302.All specimens were stored in -20℃ refrigerator until making microtome section. All sections were packed with silver sheet and stored in -20℃ refrigerator until to be stained. To detect the HLA Ⅰ (HLA ABC),HLAⅡ (HLA DR,DP,DQ) expression and endothelial cell variance before and after cryopreservation with histoimmunochemistry utilizing HLA-ABC(0736),HLA-DR(ZM-0136),CD45RO (ZM-0055)and CD34(ZM-0046),and then perform histoimmunochemistry image analysis.Results: HLA class I (HLA ABC) antigen could be demonstrated in the fresh conduit walls or on the valves(+++)-(++++).But Class 1I(HLA DR) antigen could be demonstrated in the sub-endothelial layer and in the valve matrix. After cryopreservation and thawed, HLA Class I antigen could not be demonstrated in conduits walls or on the valves(-).Class II antigen stained cells could also be seen to be weakly staining(++)-(+)in the sub-endothelial layer and in the valve matrix. CD45 antigen stained cells was in the sub-endothelial layer and in the valve matrix. These Class II staining cells were also stained by CD45 antigen and hence they could be macrophages, histiocytes or leucocytes. Endothelium was very well(++++) demonstrated in vessel walls even after cryopreservation and thawing. But endothelium could only be seen in very fresh valves(++++).In the valves the endothelium could not be seen after thawing(-). The procedure of sterilization and freezing and thawing probably destroyed the thin one celled layer of the endothelium on the valves.Conclusions: Homograft aortic and pulmonary conduits and valves from late-pregnancy fetus are immunogenic. HLA-ABC and HLA-DR antigen can be demonstrated on different components of the vessel walls and valves before and after cryopreservation. The immunogenicity is weaker after cryopreserv -ation and is not relevant with the shedding of endothelial cells.