Paraffin embedding is a foundational technique in histology, the study of tissue structure, that allows soft biological samples to be hardened for examination under a microscope. This process transforms delicate, watery tissue into a solid block, which is then sliced into incredibly thin sections, typically between 4 and 6 micrometers thick, using a specialized instrument called a microtome. These thin slices allow light to pass through and stains to effectively highlight cellular details for diagnostic purposes. Without this embedding step, the tissue would tear and collapse when faced with the microtome blade.
Preparing the Tissue for Embedding
The tissue preparation phase is necessary because the final embedding medium, paraffin wax, is water-repellent and cannot mix with the water naturally present in the tissue. The first step is fixation, often achieved by soaking the sample in a solution like 10% neutral buffered formalin. Fixation preserves the tissue structure by halting degradation and hardening the proteins. This process is typically performed for 12 to 48 hours, depending on the size of the specimen.
Following fixation, the sample must undergo dehydration to remove all residual water. This is accomplished by immersing the tissue sequentially in a series of ascending concentrations of alcohol, such as ethanol, starting from 70% and progressing up to $100\%$. Using graded concentrations prevents the tissue from shrinking or distorting excessively. Alcohol is miscible with water, allowing the water molecules within the cells to be gradually replaced.
The final preparatory step before embedding is clearing, where the alcohol is removed from the tissue. Since alcohol is not miscible with molten paraffin wax, a third solvent must be introduced that can mix with both the alcohol and the wax. Xylene is the most common clearing agent used, and the tissue is soaked until it becomes translucent, a sign that the alcohol has been fully replaced. Incomplete clearing will compromise the next step, as residual alcohol prevents the paraffin wax from fully penetrating the tissue.
Wax Infiltration and Block Casting
Once cleared, the tissue is ready for infiltration, the process where the clearing agent is replaced by molten paraffin wax. This step is often performed within an automated tissue processor that controls the temperature and timing of the fluid exchanges. The tissue is typically incubated in two or more changes of liquid paraffin wax, maintained just above its melting point, usually between $56^\circ \text{C}$ and $60^\circ \text{C}$. This elevated temperature ensures the wax remains liquid and can permeate the microscopic spaces within the tissue structure.
The next phase is block casting, which involves transferring the infiltrated tissue into a mold to form the final block. The tissue is carefully removed from the cassette and placed into a small metal mold, which is then filled with fresh molten paraffin. Speed is important because the wax will solidify quickly once it cools, which can prevent proper orientation of the sample. The mold is then topped with the tissue cassette, which acts as a base and label holder for the finished block.
The final block is rapidly cooled, often on a cold plate, to ensure the wax solidifies with a uniform, stable crystalline structure. Controlled cooling helps prevent the formation of artifacts and ensures the block has the firmness and consistency required for thin sectioning. The paraffin block encases the tissue in a rigid medium, providing support to withstand the mechanical stress of the microtome blade.
Tissue Orientation
The physical placement of the tissue within the mold, known as orientation, influences the accuracy of the resulting diagnosis. If the tissue is misaligned, the final thin section may fail to display the relevant anatomical structures, rendering the sample useless for analysis. The correct plane of section must be determined based on the tissue type to ensure that the pathologist sees the intended structures.
For instance, tubular or layered specimens, such as blood vessels, skin, or fallopian tubes, have specific orientation requirements. Skin samples must be positioned so that the outermost layer (the epidermis) faces the bottom of the mold, allowing the entire depth of the sample to be visible in a single section. Similarly, a small tubular structure must be embedded to yield either a true transverse (cross-section) or a true longitudinal view, depending on the diagnostic need. This requires careful manual placement with warm tools before the wax sets.
Ensuring Quality and Troubleshooting
Several common issues can compromise the integrity of the final paraffin block. One frequent problem is tissue shrinkage or hardening, which makes sectioning difficult and can distort cellular morphology. This typically occurs due to over-fixation or over-processing, such as leaving the tissue in the dehydration or clearing agents for too long. This excessive exposure extracts water and lipids.
Another issue is incomplete infiltration, which leaves the block with soft or mushy spots that will crumble during sectioning. This points to a failure in the preceding steps, such as incomplete dehydration (residual water prevents wax penetration) or incomplete clearing (alcohol remains). To resolve this, processing times and reagent concentrations must be carefully monitored. The quality of the paraffin wax, which should have a melting point around $56^\circ \text{C}$ to $58^\circ \text{C}$, should be verified, and air bubbles next to the tissue minimized during the casting process.