title: Detection of complement-fixing and non-fixing antibodies specific for endothelial precursor cells and lymphocytes using flow cytometry.
abstract: Donor human leukocyte antigen (HLA)-specific antibodies (Abs) with the ability to activate complement are associated with an increased risk of early Ab-mediated rejection (AMR) of kidney allografts. In recent years, also non-HLA Abs-binding endothelial cells have been shown to elicit early AMR. Donor-specific anti-endothelial cell Abs escape detection in the pre-transplant evaluation if only lymphocytes are used as target cells in crossmatch tests. We addressed whether endothelial precursor cells (EPCs) could be used for detection of complement-fixing as well as non-fixing Abs and if complement factor and immunoglobulin G (IgG) deposition on co-purified T and B cells correlated to the outcome of the T- and B-cell complement-dependent cytotoxicity assay. Deposition of complement factors C3c and C3d, but not C1q nor C4d, were detected on EPCs and lymphocytes upon incubation with HLA Ab-positive sera. There was a correlation between the amount of C3c deposition and IgG binding on EPCs (R(2) = 0.71, P = 0.0012) and T cells (R(2) = 0.74, P = 0.0006) but not for B cells (R(2) = 0.34, P = 0.059). The specificity and sensitivity for C3d deposition on endothelial precursor cell crossmatch (EPCXM) T cells vs the T complement-dependent cytotoxicity (CDC) assay were 69% and 72%, respectively. The EPCXM B-cell C3d assay had considerably lower sensitivity (39%) than the B CDC assay. Altogether, this novel assay based on the detection of complements factors on EPCs and lymphocytes by flow cytometry may widen the diagnostic repertoire and thereby improve the clinical management of patients undergoing kidney transplantation.
authors.affiliation: Division of Clinical Immunology and Transfusion Medicine, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden.
authors.forename: A
authors.initials: A
authors.lastname: AlMahri
authors.forename: J
authors.initials: J
authors.lastname: Holgersson
authors.forename: M
authors.initials: M
authors.lastname: Alheim
mesh_terms: D000918:Antibody Specificity; D001402:B-Lymphocytes / Q000737:chemistry / Q000276:immunology*; D002454:Cell Differentiation; D015922:Complement C1q / Q000276:immunology; D015932:Complement C3c / Q000276:immunology; D015933:Complement C3d / Q000276:immunology; D015935:Complement C4b / Q000276:immunology; D003601:Cytotoxicity Tests, Immunologic; D042783:Endothelial Cells / Q000737:chemistry / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods*; D006084:Graft Rejection / Q000175:diagnosis / Q000276:immunology*; D006680:HLA Antigens / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007518:Isoantibodies / Q000032:analysis* / Q000276:immunology; D016030:Kidney Transplantation / Q000276:immunology*; D008297:Male; D010446:Peptide Fragments / Q000276:immunology; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000737:chemistry / Q000276:immunology*; D014184:Transplantation, Homologous
chemical_list: D006680:HLA Antigens; D007074:Immunoglobulin G; D007518:Isoantibodies; D010446:Peptide Fragments; D015922:Complement C1q; D015932:Complement C3c; D015933:Complement C3d; D015935:Complement C4b; C032261:complement C4d