Optimization of Estrogen Receptor Analysis by Immunocytochemistry in Random Periareolar Fine-Needle Aspiration Samples of Breast Tissue Processed as Thin-Layer Preparations

Abstract

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.

Reduction in proliferation with six months of letrozole in women on hormone replacement therapy

“…The ER protocol had been optimized for specimens obtained by RPFNA, using antigen retrieval for 2 min at 90°C with a Biocare nuclear decloaker and using a 0.01% glucose oxidase blocking reagent applied for 30 min at 37°C [27]. ER was manually scored by two readers assessing 100 cells from a hyperplastic duct exhibiting ER staining.…”

“…As efficacy endpoints we assessed several risk biomarkers including cytomorphology in cells obtained by random periareolar fine needle aspiration (RPFNA) [24], Ki-67 and ER in hyperplastic epithelium [25][26][27], mammographic breast density [28], serum estradiol, testosterone [29], IGF-1, and IGFBP-3 [30]. Our primary endpoint was change in Ki-67.…”

Epigenetic regulation of transcription factor promoter regions by low-dose genistein through mitogen-activated protein kinase and mitogen-and-stress activated kinase 1 nongenomic signaling

“…The tissue slides were deparaffinized, rehydrated, peroxidase blocked, and antigen retrieved [38]; then the tissues were incubated with specific primary antibodies (overnight at 4 °C): anti-phospho-MSK1 and anti-H3S10ph (mentioned earlier in the Western blotting section), or with non-immune rabbit serum (negative control) (Jackson Immuno Research) at the same concentration as the primary antibodies. The secondary antibody (1 h at r.t., Vector Laboratories), label (Vector) and chromogen, 3,3’ diaminobenzidine tetrahydrochloride (DAB) (Dako) were applied.…”

Estrogen Receptor Expression in Benign Breast Ductal Cells Obtained from Random Periareolar Fine Needle Aspiration Correlates with Menopausal Status and Cytomorphology Index Score

“…Slides were then placed in glucose oxidase blocker at 37°C for 30 min [12]. After immunostaining for ER, slides were rinsed in distilled water and counter stained with hematoxylin (only slides containing >100 epithelial cells were utilized).…”