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
Introduction
Anatomic pathology-based assays are increasing in their complexity and relevance as the field of personalized medicine progresses. As diagnostic tests are developed for specific biomarkers, pathologists and oncologists have the potential to test the same specimen with multiple assays to determine the best course of therapy. Formalin-fixed, paraffin embedded (FFPE) tissue specimens, when sectioned and placed onto glass slides and tested with specific stains and assays, enable pathologists to observe histomorphology and specific molecular characteristics of tumor tissue and individual tumor cells. Therefore, pre-analytical conditions must preserve important histomorphological features and subcellular components, including antigens and nucleic acids, the targets of stains and assays. However, fixatives that produce the best histomorphological appearance in tissue are not necessarily optimal for preserving the molecular state of the cells. A balance between these functions in the chemical composition of tissue fixatives is required for current histopathological practices.
In addition to type of fixative [1], [3], [4], [5], [6] studies have determined that other pre-analytical factors are critical for appropriate staining results for ISH and IHC-based assays. These include the time from excision to fixation and duration of fixation [1], [7], [8], [9], [10], [11], [12], [13], [14], [15] the volume of tissue to the volume of fixative [1], [9], and post-processing factors for specific specimen types [16]. Clearly, standardization of pre-analytical processing is required to obtain optimal, accurate and reproducible results from diagnostic assays. In recognition of the importance of this idea, the American Society of Clinical Oncologists/College of Anatomical Pathologists (ASCO/CAP) recently published their recommendations for several factors related to standardization of HER2 testing in breast cancer patients, including pre-analytical processing. For both IHC and ISH-based assays, the recommendation is for 10% neutral-buffered formalin (NBF) for at least 6 h, with a maximum of 48 h [1]. Studies have verified that strict implementation of these guidelines is possible (even in large reference laboratories). Such standardization significantly decreases the number of inconsistent/inconclusive test results for breast marker IHC assays and FISH testing [2].
We have expanded these studies and investigated the impact of a range of specific pre-analytical processing factors on the staining results from in situ hybridization assays. To control the variables examined, we used the human breast carcinoma cell line, MCF7, generated as xenograft tumors in mice. We analyzed the effects of varying type and time of fixation on H&E staining as well as staining with a newly developed, fully automated dual color, dual hapten HER2 ISH (Dual ISH) assay, and an existing HER2 FISH assay. Specifically, we incubated the MCF7 tumors in commonly used fixatives including 10% neutral-buffered formalin (NBF); zinc (Zn) formalin; alcoholic formalin; Prefer (a synthetic formalin substitute); Davidson’s AFA and Bouin’s (fixatives with formalin and acids); for a range of times, varying from 1 to 120 h. In addition, we analyzed the impact of varying the thickness of cut FFPE specimens (ranging from 2 to 8 μm) on ISH staining results, using MCF7 tumors and human breast carcinoma specimens that span the dynamic range of HER2 gene status. Finally, we examined the effects of incubating formalin-fixed breast carcinoma specimens in decalcifying solutions, which is performed prior to staining tumor samples from bone. (Some cancers may metastasize to bone and there is a need to test them with various stains and molecular assays.) Our results underscore the importance of implementing a standardized pre-analytical processing system in pathology laboratories to ensure consistent and appropriate staining results for molecular diagnostic tests.
Section snippets
MCF7 cell line growth and preparation for injection into SCID mice
MCF7 cells (P/N HTB-22, ATCC, Manassas VA) were grown in sterile filtered Eagle’s MEM with l-glutamine (MediaTech Inc., Manassas VA) supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.1 mM non-essential amino acids (NEAA) (all Hyclone, Logan UT), 0.01 mg/ml insulin (Invitrogen, Carlesbad CA), and 1.0 mM sodium pyruvate (MediaTech Inc., Manassas VA). MCF7 cells were maintained in a 5% CO2 humidified 37 °C cell culture incubator in vented T75 or T150 flasks.
Culture flasks were
Results
The first set of studies was designed to assess the impact of different fixatives and the time of fixation on staining quality of H&E, FISH, and Dual ISH assays. Since there has been increasing discussion around the need for standardization of HER2 testing in breast carcinoma [1], [17], we used the HER2 ISH assays as our test system. MCF7 xenograft tumors were used as a model because the human carcinoma cells are relatively easy to generate as tumors in SCID mice, and one tumor can be divided
Discussion
The impact of multiple pre-analytic factors, including type of fixative, time from excision to fixation, the volume of tissue to the volume of fixative, duration of fixation, post-processing factors, and the thickness of the tissue specimen cut onto the glass microscope slide, are critical for optimal staining and analysis by H&E, IHC and ISH. There have been studies that address various aspects of the pre-analytical process on various diagnostic tests, but complete data sets on the impact of
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