From the Guidelines
Histological analysis is a crucial diagnostic tool that should be performed with proper specimen handling and processing to ensure accurate results, as emphasized by the most recent and highest quality study 1. When performing histological analysis, it is essential to follow a standardized protocol to ensure consistent and reliable results. This includes:
- Collecting tissue samples and immediately fixing them in 10% neutral buffered formalin (or appropriate fixative) for 24-48 hours
- Processing the tissue through dehydration, clearing, and paraffin embedding before cutting it into thin sections (4-5 micrometers) using a microtome
- Using standard staining with hematoxylin and eosin (H&E) to visualize cell nuclei and cytoplasm/extracellular matrix, and special stains like PAS, Masson's trichrome, or immunohistochemical techniques to highlight specific cellular components The accuracy of histological analysis depends on proper specimen handling, processing quality, and the pathologist's expertise in interpreting the microscopic findings within the clinical context, as noted in 1 and 1. In cases where tissue sampling is performed, it is crucial to involve a neuropathologist or pathologist in the macroscopic evaluation of the entire resected tissue and the decision as to which parts should be taken for routine diagnostics versus deep freezing or other research purposes, as recommended in 1. The use of deep-frozen tissue samples is recommended for optimal long-term preservation of sensitive analytes, such as RNA, proteins, and metabolites, and should be stored at −80°C or even lower temperatures, as suggested in 1.
From the Research
Histological Analysis Overview
- Histological procedures aim to provide good quality sections for light microscopic evaluation of human or animal tissue changes in either spontaneous or induced diseases 2, 3.
- The process involves fixing tissues with neutral formalin 10%, embedding in paraffin, and manually sectioning with a microtome to obtain 4-5 μm-thick paraffin sections 2, 3.
Tissue Staining
- Dewaxed sections are typically stained with hematoxylin and eosin (H&E) or can be used for other purposes such as special stains, immunohistochemistry, or in situ hybridization 2, 3, 4.
- H&E stains are essential for recognizing various tissue types and morphologic changes, with hematoxylin staining nucleic acids and eosin staining proteins nonspecifically 4.
Sample Preparation and Fixation
- Formalin-fixed and paraffin-embedded (FFPE) biological tissue samples are a source of clinical and molecular information, but can pose problems for genetic material extraction due to fixation 5.
- Sample preparation and fixation are critical steps in histological techniques, requiring control of methodological steps to avoid failure and complication 6.
- The goal of sample preparation is to achieve specific detection of the antigen with a strong mark and clear preservation of histological and cytological details 6.