C4d/CD34 Co-Immunofluorescence Staining for the Evaluation of the Extent of C4d Positivity in Renal Transplants.
Kuang-Yu Jen, Thuy B Nguyen, Zoltan G Laszik. University of California San Francisco
Background: Immunofluorescence (IF) detection of the complement split product C4d along tubulointerstitial (TIS) capillaries of transplant kidney biopsies is the mainstay of diagnosing antibody-mediated rejection (AMR). The extent of C4d positivity may have significant clinical ramifications; however, precise quantitative assessment of the proportion of TIS capillaries that are C4d-positive is often difficult, if not impossible. The aim of our study is to develop a C4d/CD34 double IF stain that allows not only rapid and sensitive detection of C4d positivity, but also precise and reproducible morphometric quantitation of the fraction of C4d-positive TIS capillaries.
Design: Renal transplant biopsies with C4d-positive acute or chronic AMR (n=10) as determined by single IF staining for C4d on frozen sections were used for the study. Two biopsies negative for C4d IF were used as negative controls. Frozen sections were stained with a mixture of polyclonal rabbit anti-C4d (ALPCO) and mouse anti-CD34 (Dako) antibodies followed by incubation with a mixture of fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit (Vector) and Texas Red-labeled horse anti-mouse (Vector) antibodies. Digital photographs of multiple fields were taken from each double-stained slide at 20x magnification for FITC and Texas Red IF. The photographs were processed using Just Another Colocalization Plugin (JACoP) for the ImageJ® software in order to calculate the extent of colocalization of C4d and CD34 positivity in the TIS capillaries. Manders coefficients M1 and M2 were used to determine the extent of colocalized staining. To assess the sensitivity of the C4d/CD34 double IF stain for C4d detection, the findings of C4d positivity in the double-stained sections were visually compared to the corresponding fields of serially-cut frozen sections stained for C4d alone.
Results: The extent and intensity of the C4d positivity was comparable in the double (C4d/CD34) and single (C4d) IF-stained sections. Colocalization of C4d and CD34 in the TIS capillaries as assessed by Manders coefficient M2 ranged from 0.17 to 0.87. A high degree of correlation was observed between various fields in the same biopsy for the extent of colocalization of C4d and CD34 positivity.
Conclusions: C4d/CD34 double IF stain on frozen sections yields rapid and sensitive detection of TIS capillary C4d positivity in renal transplant biopsies. It also allows simple, precise, and reproducible morphometric determination of the C4d-positive fraction of the CD34-positive TIS capillaries.
Category: Kidney (does not include tumors)
Wednesday, March 2, 2011 1:00 PM
Poster Session VI # 265, Wednesday Afternoon