title: Cross-validation of IFN-γ Elispot assay for measuring alloreactive memory/effector T cell responses in renal transplant recipients.
abstract: Assessment of donor-specific alloreactive memory/effector T cell responses using an IFN-γ Elispot assay has been suggested to be a novel immune-monitoring tool for evaluating the cellular immune risk in renal transplantation. Here, we report the cross-validation data of the IFN-γ Elispot assay performed within different European laboratories taking part of the EU RISET consortium. For this purpose, development of a standard operating procedure (SOP), comparisons of lectures of IFN-γ plates assessing intra- and interlaboratory assay variability of allogeneic or peptide stimuli in both healthy and kidney transplant individuals have been the main objectives. We show that the use of a same SOP and count-settings of the Elispot bioreader allow low coefficient variation between laboratories. Frozen and shipped samples display slightly lower detectable IFN-γ frequencies than fresh samples. Importantly, a close correlation between different laboratories is obtained when measuring high frequencies of antigen-specific primed/memory T cell alloresponses. Interestingly, significant high donor-specific alloreactive T cell responses can be similarly detected among different laboratories in kidney transplant patients displaying histological patterns of acute T cell mediated rejection. In conclusion, assessment of circulating alloreactive memory/effector T cells using an INF-γ Elispot assay can be accurately achieved using the same SOP, Elispot bioreader and experienced technicians in kidney transplantation.
authors.affiliation: Nephrology Department, Renal Transplant Unit, Bellvitge University Hospital, Barcelona University, Barcelona, Spain. 35830obm@comb.cat
authors.forename: O
authors.initials: O
authors.lastname: Bestard
authors.forename: E
authors.initials: E
authors.lastname: Crespo
authors.forename: M
authors.initials: M
authors.lastname: Stein
authors.forename: M
authors.initials: M
authors.lastname: Lúcia
authors.forename: D L
authors.initials: DL
authors.lastname: Roelen
authors.forename: Y J
authors.initials: YJ
authors.lastname: de Vaal
authors.forename: M P
authors.initials: MP
authors.lastname: Hernandez-Fuentes
authors.forename: L
authors.initials: L
authors.lastname: Chatenoud
authors.forename: K J
authors.initials: KJ
authors.lastname: Wood
authors.forename: F H
authors.initials: FH
authors.lastname: Claas
authors.forename: J M
authors.initials: JM
authors.lastname: Cruzado
authors.forename: J M
authors.initials: JM
authors.lastname: Grinyó
authors.forename: H D
authors.initials: HD
authors.lastname: Volk
authors.forename: P
authors.initials: P
authors.lastname: Reinke
mesh_terms: D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D058501:Enzyme-Linked Immunospot Assay / Q000379:methods*; D006084:Graft Rejection / Q000276:immunology*; D006801:Humans; D007111:Immunity, Cellular / Q000276:immunology*; D007156:Immunologic Memory; D007371:Interferon-gamma / Q000276:immunology*; D016030:Kidney Transplantation / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D007371:Interferon-gamma