OR46 A novel cellular crossmatch to detect donor HLA specific immunity

Abstract

Aim To develop a cellular crossmatch method (CXM) for evaluating donor HLA specific cellular immunity in HLA mismatched recipients. Methods HLA Typing: HLA types of all samples were obtained by high resolution typing. CXM: Irradiated donor cells were used as the donor HLA specific stimulant to co-incubate with recipient (responder) cells and Th1 (IFNγ & IL-2) or Th2 (IL-4, 5, 10) cytokine capture beads for overnight in fixed cell:bead ratio. The cells were lysed and the captured cytokines were detected by corresponding fluorescent 2nd step antibodies. The percentage of positive beads (Flowspot %) were calculated which proportionately correlated with numbers of donor specific responding cells. The MFI level correlated with the cytokine secretion capability of responding cells. Results A total 147 paired bone marrow donor and recipient samples were tested by CXM. Compared to the HLA identical group, IL-2 but not IFNγ was significantly increased in HLA-class I (CI) (P Conclusions Unlike mixed lymphocyte reaction (MLR) which detects DNA synthesis by radioisotope, the newly developed CXM is able to detect donor HLA specific immunity in responder within 24 h using standard flow cytometry. CXM detects earlier T cell activation (e.g. cytokine secretions) in a uni-or bi-directional multiplex detecting system. The method of CXM is simple, sensitive, and donor HLA specific. It potentially has wide application in both the research and clinical fields for predicting GVH/GVL in BM transplantation, cellular rejection in solid organ transplantation, and in other applications where cell–cell responses are of interest. Download : Download high-res image (207KB) Download : Download full-size image