Optimal Fixation Conditions for Immunocytochemical Analysis of Estrogen Receptor in Cytologic Specimens of Breast Carcinoma

BACKGROUND. The techniques for immunostaining estrogen receptor (ER) in cytologic specimens have varied, as have the detection rates. The authors compared various fixation methods for their effect on ER detection in cytologic smears of breast carcinoma. METHODS. Smears were prepared by gently scraping the cut surfaces of 47 resected breast carcinoma specimens and placing immediately in 1 of the following conditions: 1) a sequence of 10% formalin-methanol-acetone fixatives at -20 °C (Abbott method); 2) 10% formalin at room temperature; and 3) Carnoy's fixative at room temperature and then Papanicolaou stained (Carnoy's-Pap). Destaining of Carnoy's-Pap smears (Carnoy's-dPap) was initially attempted before ER staining. One set of smears was also air-dried for 3 minutes before using the Abbott method. Smears and corresponding tissue sections were immunostained with anti-ER antibody 6F11 using a similar protocol except for antigen retrieval, which was not initially applied on cytologic slides. All the ER-negative smears that had been fixed with 10% formalin or Carnoy's-Pap were restained after antigen retrieval. Agreement between cytologic and histologic findings was expressed by both concordance and the kappa coefficient. RESULTS. ER detection in smears processed with the Abbott method correlated best with findings from tissue samples, with an overall correlation of 91.5% (κ = 0.80). Findings from air-dried smears were less optimal (concordance, 84.4%; κ = 0.65), followed by Carnoy's-Pap (concordance, 71.4%; K = 0.45), formalin (concordance, 31%; κ = 0.05), and Carnoy's-dPap (concordance, 29.4%; κ = 0.04). Antigen retrieval converted most of the ER-negative smears to positive (18 of 20 smears in formalin and 6 of 8 smears in Carnoy's-Pap), leading to a final concordance of 93% and κ = 0.83 for both conditions. Antigen retrieval also led to stronger staining intensity without causing false positivity. CONCLUSIONS. Antigen retrieval was found to greatly improve ER immunodetectability and staining intensity in formalin-fixed and Carnoy's-Pap smears. The former may offer an alternative to the Abbott method because of its easiness and the latter can be reliably used in archival Pap-stained smears for retrospective analysis of ER. Air-drying, destaining Pap smear, and fixatior in formalin or Carnoy's-Pap without antigen retrieval are not recommended.