Tissue processing in histology transforms a soft, raw biological sample into a durable, thin slice mounted on a glass slide for detailed microscopic examination. This multi-step chemical and physical workflow is foundational to diagnostic pathology, allowing medical professionals to observe cellular architecture to identify diseases, such as cancer. The process ensures the delicate tissue is chemically preserved and physically supported, making it possible to create the ultra-thin sections necessary for visualization under a microscope.
Phase One: Preserving Cellular Structure (Fixation)
The first step in tissue processing is chemical fixation, a procedure that halts all biological activity within the sample to preserve its structure as close to the living state as possible. Fixation works primarily by preventing two processes: autolysis, or self-digestion by the tissue’s own enzymes, and putrefaction, which is decomposition caused by bacteria and fungi. A fixative must quickly penetrate the tissue and chemically stabilize its components.
The most common fixative used in routine histopathology is 10% neutral buffered formalin, which is an aqueous solution of formaldehyde gas. Formaldehyde acts by forming chemical cross-links between protein molecules, stabilizing the cellular and sub-cellular components. This cross-linking is thought to occur when the formaldehyde reacts with primary amine groups on amino acid residues, forming a methylene bridge between nearby proteins.
Proper fixation requires the tissue be trimmed to allow the fixative to penetrate fully, typically not exceeding 3 to 4 millimeters in thickness. The volume of the fixative solution should be significantly greater than the tissue volume, often a 10:1 ratio. Fixation time ranges from 6 to 48 hours, depending on the specimen’s size and density. Maintaining the correct temperature and a neutral pH minimizes tissue distortion.
Phase Two: Preparing for Support (Dehydration, Clearing, and Embedding)
Following preservation, the tissue is prepared for physical support by removing all water and replacing it with paraffin wax. This is necessary because paraffin wax is hydrophobic and immiscible with water. The first stage is dehydration, which involves immersing the fixed tissue in a series of ascending alcohol solutions, typically starting around 70% ethanol and progressing up to 100% alcohol.
The gradual increase in alcohol concentration prevents the sudden osmotic shock that would severely shrink and damage the tissue architecture. Once the tissue is fully dehydrated, the alcohol must be removed, as it is not miscible with the embedding material. This is accomplished in the next step, known as clearing, where the tissue is immersed in a solvent such as xylene.
Xylene is miscible with both the absolute alcohol and the final embedding medium. The clearing agent effectively displaces the alcohol, making the tissue appear translucent. After the alcohol is replaced, the tissue is ready for infiltration, where it is transferred into a bath of molten paraffin wax, usually heated to approximately 56 to 58 degrees Celsius.
Infiltration is the process of the liquid wax permeating and filling all the spaces within the tissue. The tissue remains in the molten wax for several hours to ensure complete penetration, providing the necessary internal support. After infiltration, the tissue is carefully oriented in a mold and covered with fresh molten wax, which is then allowed to cool and solidify, creating a firm paraffin block ready for sectioning.
Phase Three: Creating the Diagnostic Slide (Sectioning and Staining)
The hardened paraffin block, which now contains the preserved tissue, is mounted onto a specialized instrument called a microtome. The microtome uses a precisely angled blade to slice the block into extremely thin sections, typically measuring 4 to 5 micrometers thick, which is thinner than a red blood cell. As the block is repeatedly sliced, the sections adhere to one another, forming a delicate ribbon of tissue and wax.
These fragile ribbons are then floated onto the surface of a warm water bath to smooth out any wrinkles or compression that occurred during cutting. A single section is carefully picked up from the water bath onto a clean glass slide. The tissue section is colorless, so the paraffin wax must be removed with a solvent, followed by rehydration, preparing it for the final chemical step: staining.
The most common method is Hematoxylin and Eosin (H&E) staining, which provides contrast and highlights cellular structures. Hematoxylin is a basic dye that stains basophilic structures, primarily the cell nuclei, a purplish-blue color. Eosin is an acidic dye that stains eosinophilic structures, such as the cytoplasm and extracellular matrix proteins, various shades of pink or red. This differential staining creates a clear contrast between the nucleus and surrounding components, allowing pathologists to analyze the tissue’s morphology and make a diagnosis. Finally, the stained section is permanently preserved under a protective coverslip, completing the diagnostic slide.