Estrogen, progesterone, and HER‐2 receptor immunostaining in cytology: The effect of varied fixation on human breast cancer cells

The ASCO/CAP Expert Panel recommends that all invasive breast carcinomas and breast cancer recurrences be tested for ER, PR and HER‐2 expression. The guidelines for testing of surgical specimens by immunohistochemistry (IHC) are well defined, whereas they are lacking for cytological samples. We evaluated various fixation protocols for optimal receptor testing by immunohistochemistry and immunocytochemistry (ICC) of human breast cancer cell lines MCF‐7 (ER/PR positive) and SKBR‐3 (overexpressing HER‐2). The cells were fixed in 10% neutral buffered formalin or Saccomanno Fixative (SF) for various time points, and either embedded in paraffin as cell blocks or prepared as cytospins. ER and PR slides were assigned a proportion score (PS; 0–5), an intensity score (IS; 0–3) and a total score (TS = PS + IS). Standard DAKO scoring system ranging from 0 to 3+ was used for the evaluation of HER‐2 staining. Human breast cancer cells stained successfully for ER, PR and HER‐2 when fixed in formalin and prepared as cell blocks. The optimal fixation time for formalin‐fixed cells ranged from 2 to 96 hours. Cells fixed in SF from 2 to 96 hours also stained well for ER and PR. However, SF produced variable results for HER‐2 staining; particularly, SF fixation beyond 24 hours caused false negative results. The interpretation of HER‐2 staining on cytospins was not feasible irrespective of the fixative and fixation time. In summary, formalin fixation from 2 to 96 hours and preparation of cells as cell blocks produces optimal results for ER, PR, and HER‐2 testing in human breast cancer cells. Diagn. Cytopathol. 2013;41:864–870. © 2013 Wiley Periodicals, Inc.