Therefore, an accurate and reproducible method for assessing the percentage of peritubular capillary C4d positivity is greatly needed. Furthermore, positive C4d signal in other structures, such as tubular basement membranes, may mimic peritubular capillaries. This obstacle is especially evident in biopsies with significant interstitial fibrosis and tubular atrophy where the density of peritubular capillaries is uncertain. 6, 7, 8 Although the fraction of C4d-positive peritubular capillaries can be visually estimated using this method, it is not uncommon to come across situations where single-channel C4d immunofluorescence is extremely difficult to interpret. Currently, indirect immunofluorescence staining with anti-C4d antibody on frozen sections of fresh/unfixed tissues is the most widely used method and offers slightly higher sensitivity than immunohistochemical stains. In order to make any headway into tackling this issue, a reliable method for assessing the extent of C4d-positive peritubular capillaries is essential. 2, 3, 4, 5 Nevertheless, the data are limited and further evaluation with regard to long-term graft survival is needed. Some studies suggest that even focal C4d staining may have significant clinical ramifications, while other studies fail to show such outcomes. 1 However, the clinical significance of focal (<50%) peritubular capillary C4d positivity remains unclear. Diffuse C4d positivity, defined as >50% peritubular capillary involvement, is highly correlated to the presence of circulating donor-specific antibodies and is required by the Banff 07 Classification of Renal Allograft Pathology for the diagnosis of acute and chronic antibody-mediated rejection. Positive immunostaining for the complement split product C4d within peritubular capillaries is critical for the diagnosis of antibody-mediated rejection in renal allograft biopsies. C4d/CD34 double immunofluorescence provides rapid, convenient, and low-cost implementation for laboratories currently utilizing single-channel C4d immunofluorescence. 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. 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. 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. 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.
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