Optimization of estrogen receptor analysis by immunocytochemistry in random periareolar fine-needle aspiration...

Immunostaining of estrogen receptor alpha (ER) in samples of benign breast tissue obtained by random periareolar fine-needle aspiration (RPFNA) is a practical tool for breast cancer chemoprevention trials. The authors report an optimized method of ER immunostaining for use with thin-layer preparations of modified Cytolyt-fixed benign breast tissue acquired by RPFNA. Samples of benign breast tissue and MCF-7 controls processed as thin-layer preparations were tested for the effects of antibody titer, antigen retrieval temperature (90 degrees or 115 degrees C), buffer (20% nuclear decloaker, pH 9.3; 10 mM citrate buffer, pH 6), and blocking solution (0.01% glucose oxidase or 0.3% H2O2) on ER immunostaining. The prevalence of positively stained breast epithelial cells, mean intensity of ER staining, and composite immunostaining score were evaluated for effect of immunostaining protocol. RPFNA samples and MCF-7 cells processed using nuclear decloaker and low-temperature antigen retrieval had more ER-positive cells (P<0.0001) and increased mean staining intensity and weighted staining indices (P<0.05) compared with samples prepared with citrate buffer and high-temperature antigen retrieval. Glucose oxidase increased ER-positive cells in comparison to hydrogen peroxide (P<0.04) when combined with low-temperature antigen retrieval and the use of nuclear decloaker. Staining was negative for all non-immune controls regardless of protocol. The combination of low-temperature antigen retrieval, diluted commercial nuclear decloaker solution, and a glucose oxidase blocking step yielded optimal ER immunostaining for thin-layer preparations of benign breast tissue harvested by RPFNA.