Influence of Fixation, Antibody Clones, and Signal Amplification on Steroid Receptor Analysis

Abstract

: Various factors influence analysis of steroid receptors, including tissue processing, fixation, and antibody clones used. Furthermore, the detection methods, that is, use of epitope retrieval by microwave heating and signal amplification, may affect the reliability of the results. Eleven different fixation/tissue-processing methods as well as different commercial antibodies for the estrogen-and progesterone-receptors and the tyramine amplification technique were evaluated for optimizing the staining procedures. Moreover, the results from the optimal immunohistochemical procedure were compared with results obtained using a biochemical dextran-coated charcoal assay in 88 breast carcinomas from postmenopausal patients. The four antibody clones tested (1D5, 6F11, 1A6, and antiserum) revealed different sensibility to formalin, either favoring shorter or longer periods of fixation. Overall, a delay in the onset of fixation showed the worst results, whereas overfixation for up to 4 days or pH of formalin had little influence. Using the new tyramine technique it was possible to reduce costs per slide considerably by increasing antibody dilution. Comparison of the biochemical assay with optimized immunohistochemistry in the 88 cases investigated showed an overall concordance of 98.9%. Our study suggests an optimized immunohistochemical procedure that can replace the biochemical “gold standard” if standardization of tissue processing is maintained.