Multiplex Immunofluorescence Analysis of Infiltrating Mononuclear Cell Subsets in Acute T-cell-mediated Rejection and Bk Virus-Associated Nephropathy in The Allograft Kidney

Abstract Transplant rejection has been diagnosed by mononuclear cell infiltration patterns light microscopically. Although infiltration involves different types of mononuclear cells, analysis of the rejection-associated variation was difficult by usual immunohistochemistry; one antibody staining on each slide and laborious cell counting. The authors applied the newly developed multiplex immunofluorescence staining(MIF) and image analysis system to overcome those disadvantages. By using formalin-fixed, paraffin-embedded slides of acute T-cell–mediated rejection (TCMR, 9 cases) and BK virus-associated nephropathy (BKVAN, 5 cases), the immunologic analysis was done because both had similar mononuclear cell infiltration patterns histologically. Antibodies to CD4, CD8, CD20, CD68, Foxp3, and cytokeratin were used.The cellular subsets were similar to each other. CD68+ cells were dominant in the cortical interstitium. However, by using this method, we could find differences in cellular distributions in the area by area. In the medullary rays, albeit the location being in the cortex, cellular subsets were similar to those of the medulla. In areas surrounding large vessels thought of as non-diagnostic areas, the CD20+ cells were dominant. This MIF could use as an ancillary method of diagnosis and follow up the cellular changes during rejection or treatment. Further large samples with rejection condition-matched studies should be followed.