C4d/CD34 double-immunofluorescence staining of renal allograft biopsies for assessing peritubular capillary C4d positivity

Immunofluorescence detection of the complement split product C4d along peritubular capillaries in renal allograft biopsies is the mainstay for the diagnosis of antibody-mediated rejection. The extent of peritubular capillary C4d positivity may have significant clinical ramifications; however, peritubular capillary density in the renal cortex is often difficult to assess with single-channel immunofluorescence. In this study, we report a C4d/CD34 double-immunofluorescence staining protocol for renal allograft frozen sections that allows rapid and sensitive detection of C4d positivity, as well as improved accuracy in estimating the C4d-positive fraction of peritubular capillaries. In addition, this method aids in determining whether C4d-positive structures correspond to peritubular capillaries or whether they represent common mimics of peritubular capillaries such as tubular basement membranes. C4d/CD34 double immunofluorescence provides rapid, convenient, and low-cost implementation for laboratories currently utilizing single-channel C4d immunofluorescence.