Abstract
BackgroundThe exact composition and localization of the inflammatory burden during allograft rejection is difficult to analyse on the same biopsy slide. We tested the feasibility of detecting four distinct markers in a same paraffin-embedded tissue section from human kidney allograft rejection by using an innovative process of multiplex immunofluorescence. MethodsKidney allograft biopsies from 20 antibody-mediated rejection, 20 T cell-mediated rejection and five non rejection were labelled against NKp46, CD163, CD3, and CD34 respectively for NK cells, macrophages, T cells and endothelial cells. Images were scanned and cells were automatically quantified and their extra- or intravascular location determined. Conventional immunohistochemistry against NKp46 with manual quantification and real time quantitative polymerase chain reaction for evaluation of the relative messenger ribonucleic acid (mRNA) expression levels of NK, T cell and macrophage transcripts were simultaneously performed. ResultsMultiplex immunofluorescence cell quantification was strongly correlated to manual quantification by immunohistochemistry (r=0.91, P<0.001) and to mRNA expression levels (r>0.46, P<0.021). T cells and macrophages were the two predominant populations involved in rejection (48.0±4.4% and 49.3±4.4% in antibody-mediated rejection; 51.8±6.0% and 45.3±5.8% in T cell-mediated rejection respectively) despite an important heterogeneity in the composition of the inflammatory burden. NK cells constituted a rare population for both T cell-mediated rejection (2.9±0.6%) and antibody-mediated rejection (2.7±0.7%). The intravascular compartment was mainly composed of T cells, including during antibody-mediated rejection. However, NK cells and macrophages densities were significantly higher in capillaries during antibody-mediated rejection. ConclusionMultiplex immunofluorescence staining is a reliable technology allowing studying the exact composition and localization of the inflammatory burden during kidney allograft rejection.
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