Optimal antigen retrieval for ethanol‐fixed cytologic smears

BACKGROUND: Antigen retrieval, a crucial technique for immunostaining, is often carried out on formalin-fixed, paraffin-embedded (FFPE) tissue sections. The role of antigen retrieval in immunostaining of ethanol-fixed smears remains unclear. The authors evaluated the effects of 2 common antigen retrieval procedures, heat-induced antigen retrieval and protease-induced antigen retrieval, for immunostaining using a broad panel of antibodies. METHODS: Papanicolaou-stained ethanol-fixed smears from 36 surgical specimens were immunostained with 43 antibodies. Three widely used heat-induced antigen retrieval solutions, namely, citrate buffer (pH 6.0 and pH 7.0) and ethylenediaminetetraacetic acid solution (pH 8.0) for heat-induced antigen retrieval, and pronase were used. The staining results were compared between the ethanol-fixed smears and the corresponding FFPE tissue sections. RESULTS: Heat-induced antigen retrieval was essential for all the 9 antibodies examined against nuclear antigens, and for 7 of 26 antibodies against cytoplasmic and cell membrane antigens. Superior results were obtained using lower-pH heat-induced antigen retrieval solutions for ethanol-fixed smears than was the case for FFPE tissue sections; use of citrate buffer (pH 6.0) was optimal for most antibodies. For 17 antibodies against cytoplasmic/cell membrane antigens, satisfactory results were obtained even without antigen retrieval on the ethanol-fixed smears, whereas antigen retrieval was necessary for detection on the FFPE tissue sections. Protease-induced antigen retrieval frequently exerted deleterious effects on ethanol-fixed smears. Despite antigen retrieval, detection of 2 lymphocytic markers failed on ethanol-fixed smears. This limitation was overcome by heat-induced antigen retrieval on formalin vapor-fixed smears. CONCLUSIONS: In ethanol-fixed smears, most of the antibodies can be immunostained successfully without antigen retrieval treatment or mild heat-induced antigen retrieval using citrate buffer (pH 6.0). The optimal antigen retrieval condition for each antibody must be individually determined.