Delay to Formalin Fixation Alters Morphology and Immunohistochemistry for Breast Carcinoma

Background: Delay to formalin fixation (DFF) also known as cold ischemia has been known to have an adverse effect on breast biomarkers. The aim of the study was to further evaluate the effect of DFF on tumor histomorphology and immunohistochemistry. Materials and Methods: Ten palpable invasive breast carcinomas were resected and underwent immediate gross evaluation. For each case, the procured tumor was divided into 8 equal parts and consecutively fixed after 0, 10, and 30 minutes, I, 2, 4, and 8 hours; 1 section was kept in saline and stored overnight at 4 C. Stains normally used in breast pathology including epidermal growth factor receptor, Ki-67, p53, AE 1/3, CAM-5.2, cytokeratin (CK)7, CK 14, CK5/6, epithelial membrane antigen, E-cadherin, gross cystic disease fluid protein 15, and mammaglobin were studied. The change was considered significant if it had any pathologic or clinical consequence. Results: DFF had spurious effect on 9 of 10 cases. However, significant alteration that affected the histomorphologic interpretation was observed in 3 cases. Significant immunohistochemistry alteration was seen only in the membranous markers (epidermal growth factor receptor and E-cadherin). Although the quality of the cytoplasmic markers was affected, the interpretation (positive vs. negative) was not affected by DFF. Ki-67 had minimal but insignificant change. Generally, these changes occurred at or after 2-hour mark. Conclusions: Therefore, specimens should be fixed within 1 hour from tissue harvesting and not stored without fixation overnight.