The impact of pre-analytical processing on staining quality for H&E, dual hapten, dual color in situ hybridization and fluorescent in situ hybridization assays

Abstract

With the advent of personalized medicine, anatomic pathology-based molecular assays, including in situ hybridization (ISH) and mRNA detection tests, are performed routinely in many laboratories and have increased in their clinical importance and complexity. These assays require appropriately fixed tissue samples that preserve both nucleic acid targets and histomorphology to ensure reliable test results for determining patient treatment options. However, all aspects of tissue processing, including time until tissue fixation, type of fixative, duration of fixation, post-fixation treatments, and sectioning of the sample, impact the staining results. ASCO/CAP has issued pre-analytical guidelines to standardize tissue processing for HER2 testing in breast carcinoma specimens: 10% neutral-buffered formalin (NBF) with a fixation time from at least 6 to 48h [1]. Often, this recommendation is not followed to the detriment of staining results [2]. In this paper, we used a human breast carcinoma cell line (MCF7) generated as xenograft tumors as a model system to analyze the effects of different pre-analytical conditions on ISH staining. We performed H&E, FISH and dual colorimetric HER2 ISH assays using specimens fixed across a range of times in six different commonly used fixatives. Additionally, we investigated the effects of varying tissue section thickness, which also impacted the quality of ISH staining. Finally, we evaluated the effects of three different decalcifying solutions on human breast specimens, typically a treatment that occurs post-fixation for evaluating metastases to bone. The results indicate that time and type of fixation treatment, as well as appropriate tissue thickness and post-fixation treatment, all contribute to the quality of ISH staining results. Our data support the ASCO/CAP recommendations for standardized tissue processing (at least 6h in formalin-based fixatives and 4μm section thickness) and indicate that certain fixatives and post-fixative treatments are detrimental to molecular staining results.