Multicolor Flowcytometric Measurement of Alloantigen-Specific Antibody Secreting Cells in Highly Sensitized Patients in Renal Transplantation.

Abstract

Not all pretransplantation donor-specific antibodies (DSA) cause worse graft outcome. The increase of DSAs post-transplantation can be more important marker than the level of DSA in pretransplant sera. The assessment of sensitized cells which can secrete DSA is needed. Our aim was to develop an assay for antibody secreting cells (ASC) by multicolor flowcytometry in short-term culture of peripheral blood. Five highly-sensitized patients who were awaiting or received renal transplantaion and 3 non-sensitized patients were included. PRA screening and identification were performed with LIFECODES Life-Screen Deluxe and Class I and Class II ID kits (Gen-probe). Peripheral blood mononuclear cells were seeded with CpG 2006: 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'), (Bioneer, Korea), 500 ng/ml human CD40 Ligand (Sigma), and 50 ng/ml IL-21 (PeproTech). Lymphocyte lysates of HLA types (corresponding to specificities detected in the serum of the patient to be tested) were added to each test cells. After 72 hrs, ELISPOT analysis with Human IgG ELISpotPlus kit (Mabtech, Sweden) and flowcytometric analyses with Anti-CD19-PE, CD27-PerCP-CY5.5, CD38-APC, IgG-FITC antibodies (BD Pharmingen) were performed. The proportion of ASC was significantly increased when stimulated with lymphocyte lysate of donor specific HLA antigen than controls in highly sensitized renal transplantation patients. In non-sensitized patients, there was no difference between culture with lymphocyte lysate and controls. The absolute number of ASC was also significantly increased when stimulated with lymphocyte lysate of donor specific HLA than controls in highly sensitized patients. The number of total recovered cells and the number of ASC in ELISPOT analysis stimulated with lymphocyte lysate of donor specific HLA showed tendency of increase but didn't reach a statistical significance compared to controls in highly sensitized patients. Three day-culture condition using peripheral blood in highly sensitized patients can differentiate ASC with flowcytometric analysis. It can be applied for future study to see the increase of ASC can predicts renal tranplantation outcome.