title: Ovine panel reactive antibody assay of HLA responsivity to allograft bioengineered vascular scaffolds.
abstract: BACKGROUND
  Increasing evidence implicates immune response as a contributing factor in the failure of allograft valve transplants. Increases in panel reactive antibodies have been identified in human subjects. To correlate these responses with novel preimplantation processing methods to reduce cellularity, both a relevant panel reactive antibody assay and a chronic implantation animal model are necessary. We modified a human flow cytometric panel reactive antibody assay for ovine model use to detect antibody responses to residual antigen-loading decellularized scaffolds engineered from pulmonary artery tissue.
  
  
  METHODS
  A clinical panel reactive antibody assay was modified with anti-sheep antibodies. Dimethyl sulfoxide cryopreserved (n = 4) and decellularized scaffolds (n = 8) fashioned as patches from pulmonary arteries were implanted for study. Fresh (nonprocessed) tissue implants were used as positive controls (n = 2), and sham-treated animals were used as negative controls (n = 2). Baseline, 10-week, and 20-week blood samples were assayed for panel reactive antibody levels. Immunohistochemistry with anti-major histocompatibility complex antibodies were performed on preimplantation scaffolds.
  
  
  RESULTS
  Chronic implants of fresh tissue stimulated strong panel reactive antibody responses. Classically cryopreserved tissues provoked modest panel reactive antibody responses to major histocompatibility complex I antigen and no response to major histocompatibility complex II antigen. Decellularized tissue scaffolds provoked minimal to no panel reactive antibody responses to either major histocompatibility complex I or II antigen. Immunohistochemistry correlated with the panel reactive antibody results by identifying significant amounts of major histocompatibility complex I and II in fresh tissue, reduced antigen staining in cryopreserved control tissues, and minimal amounts in decellularized tissues.
  
  
  CONCLUSIONS
  These studies with an ovine modified panel reactive antibody assay confirmed minimal immune allosensitization to transplanted decellularized tissue patches. Qualifying criteria for putative tissue-engineered scaffolds should include minimal recipient panel reactive antibody response.
authors.affiliation: Division of Cardiothoracic Surgery and the Collis Surgical Research Laboratory, Brown University, Providence, RI, USA.
authors.forename: Ara
authors.initials: A
authors.lastname: Ketchedjian
authors.forename: Paula
authors.initials: P
authors.lastname: Kreuger
authors.forename: Howard
authors.initials: H
authors.lastname: Lukoff
authors.forename: Elliot
authors.initials: E
authors.lastname: Robinson
authors.forename: Alyce
authors.initials: A
authors.lastname: Linthurst-Jones
authors.forename: Katrina
authors.initials: K
authors.lastname: Crouch
authors.forename: Lloyd
authors.initials: L
authors.lastname: Wolfinbarger
authors.forename: Richard
authors.initials: R
authors.lastname: Hopkins
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D001698:Biomedical Engineering; D001808:Blood Vessels / Q000473:pathology / Q000637:transplantation*; D002315:Cardiopulmonary Bypass; D015925:Cryopreservation; D004195:Disease Models, Animal; D005434:Flow Cytometry; D006084:Graft Rejection; D006085:Graft Survival; D006680:HLA Antigens / Q000276:immunology*; D006650:Histocompatibility Testing; D007150:Immunohistochemistry; D011336:Probability; D034561:Sheep, Domestic; D014181:Transplantation Immunology; D014184:Transplantation, Homologous
chemical_list: D006680:HLA Antigens