Comparison of C4d immunostaining methods in renal allograft biopsies

Immunostaining of renal allograft biopsies for C4d deposition has become an important diagnostic tool in the recognition of humoral-mediated graft rejection. The majority of studies have been performed on frozen tissue sections with one of several commercially available antibody reagents. However, only a single small series that compared reagents or methods, including staining of formalin-fixed, paraffin-embedded tissue, has been published. Two different staining methods in 138 renal allograft biopsies were compared directly: A mAb (Quidel, San Diego, CA) on frozen tissue sections with indirect immunofluorescence (IF) and a polyclonal antibody (Biomedica Gruppe, distributed by ALPCO, Windham, NH) applied to formalin-fixed, paraffin-embedded tissue with immunohistochemical (IHC) detection. An initial data set of 107 consecutive cases showed complete agreement between staining methods in 104 (97%) cases. Overall, nine of 107 cases were positive with one or both methods, representing 8.4% of all allograft biopsies tested, 15% of clinically indicated biopsies, and 24% of biopsies with a histologic diagnosis of acute cellular rejection. A second set of 31 cases included 17 cases that were positive by either method, with concordance in 29 of 31 cases. Combining the two data sets, the overall specificity of the IHC method compared with IF was 98%, and sensitivity was 87.5%. Direct comparison demonstrates that IHC staining of formalin-fixed, paraffin-embedded tissue with anti-C4d polyclonal antibody has acceptable sensitivity and specificity, as compared with IF staining of frozen tissue with the Quidel mAb.