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

Mod Pathol. 2012 Mar;25(3):434-8. doi: 10.1038/modpathol.2011.168. Epub 2011 Oct 28.


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.

MeSH terms

  • Antigens, CD34 / metabolism*
  • Biomarkers / metabolism*
  • Biopsy
  • Capillaries / metabolism*
  • Complement C4b / metabolism*
  • Fluorescent Antibody Technique / methods*
  • Frozen Sections
  • Humans
  • Kidney Transplantation*
  • Kidney Tubules / blood supply*
  • Microscopy, Fluorescence
  • Peptide Fragments / metabolism*
  • Transplantation, Homologous


  • Antigens, CD34
  • Biomarkers
  • Peptide Fragments
  • Complement C4b
  • complement C4d