Optimal antigen retrieval for ethanol‐fixed cytologic smears

Abstract

BACKGROUND: Antigen retrieval, a crucial technique for immunostaining, is often carried out on formalin-fixed, paraffinembedded (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 proteaseinduced 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 ethanolfixed 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. Proteaseinduced 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. Cancer (Cancer Cytopathol) 2012;120:167À76. V C 2012 American Cancer Society.KEY WORDS: ethanol-fixed smear, formalin vapor-fixed smear, immunocytochemistry, heat-induced antigen retrieval, protease-induced antigen retrieval.

When Tissue Antigens and Antibodies Get Along

“…166 HIER using citrate buffer pH 6.0 is necessary for most nuclear antigens on ethanol-fixed cytologic smears; for some cytoplasmic membrane and cytoplasmic antigens, it enhances the immunoreaction. 80 Few cytoplasmic membrane antigens require HIER with higher pH buffers. 80 Some laboratories perform HIER irrespective of the fixation type.…”

“…80 Few cytoplasmic membrane antigens require HIER with higher pH buffers. 80 Some laboratories perform HIER irrespective of the fixation type. 435 Enzymatic AR is much less commonly used than HIER in ICC.…”

Advanced Diagnostic Techniques

“…AFCPs without AR performed similarly to cell blocks with AR in many cases; however, for some tests, particularly those detecting nuclear antigens, AR improved the reactivity of AFCPs (Ikeda et al, 2011). Few cytoplasmic membrane antigens require HIER with higher pH buffers (Denda et al, 2012). Few cytoplasmic membrane antigens require HIER with higher pH buffers (Denda et al, 2012).…”

“…Unfortunately, the wide range of fixation procedures makes standardization of AR procedures among laboratories very difficult; each laboratory must optimize the procedure for each antigen (Kirbis et al, 2011;Shtilbans et al, 2005). HIER using citrate buffer pH 6.0 is necessary for most nuclear antigens on ethanol-fixed cytologic smears; for some cytoplasmic membrane and cytoplasmic antigens, it enhances the immunoreaction (Denda et al, 2012). HIER using citrate buffer pH 6.0 is necessary for most nuclear antigens on ethanol-fixed cytologic smears; for some cytoplasmic membrane and cytoplasmic antigens, it enhances the immunoreaction (Denda et al, 2012).…”

“Clinical” cytology for endoscopists: A practical guide

“…Another disadvantage is that alcohol fixated smears are harder to use for further immunocytological staining methods. [24] The easier method to apply is May-Gruenwald-Giemsa (MGG) staining. This staining method is currently used as a standard staining method in hematology for blood – and bone marrow – smears and is routinely available in every laboratory.…”