There are numerous steps involved in preparing tissue samples for histology. Fixed tissue cannot simply be taken and embedded. This article focuses on the in-between stage of processing tissue for histology. These six steps occur between tissue fixation and the embedding or sectioning processes.
Six Steps in Tissue Processing for Histology
Step 1: Labeling Cassettes
After fixation, tissue samples are transferred to a tissue cassette. These come in a variety of sizes. They hold and protect tissue during the process. Once the embedding stage has been reached, the cassette lid is removed. The primary part of the cassette forms a base for the paraffin wax block. Cassettes have to be properly labeled either by hand or using a cassette labeling machine. The three steps in processing tissue include dehydrating, clearing, and infiltrating. Each of these steps involves diffusing the solution into tissue and dispersing the previous solution.
Step 2: Carousel Machine
Dedicated tissue processing machines carry out tissue processing for histology in most of today’s modern labs and institutes. Older machines were designed similarly to a carousel containing a cage where the cassettes were placed. Inside the carousel, glass beakers contain solvents and solutions that ensure the tissue is dehydrated, cleared, and ready for embedding in paraffin wax. The carousel agitates the cage in each solution and continues moving through the various solutions in the dehydration and clearing method. Modern processors contain a chamber that holds specimens while the various solutions are pumped in and out of the chamber. Generally, the process takes about six hours and most labs set it up to run overnight.
Step 3: Remove the Water
The tissue samples need to be dehydrated first. The water may be bound to the tissue or free. Paraffin wax is hydrophobic, so the water has to be removed before the tissue can be infiltrated with wax. The water is carried out by immersing the tissue into a series of ethanol solutions. The series of solutions contain increasing concentrations of ethanol until a 100% water-free alcohol is achieved. The increasing concentrations ensure the water in the tissue is slowly replaced by alcohol and to avoid distortion of the tissue. During the process, some components of the cell are removed.
Step 4: Ethanol Won’t Mix with Wax
Even though the tissue reaches the final stage of dehydration in ethanol, it cannot proceed directly to wax embedding. Ethanol and wax will not mix. Tissue clearing is necessary. Clearing refers to the solvents used. They have a high refractive index. When tissue is immersed in the solvents, it becomes clear and transparent.
Step 5: Clearing Agents
Xylene is a solvent that is used in the intermediate stage. This clearing agent is used since it is miscible with both paraffin wax and ethanol. After dehydration, the tissue is immersed in xylene solutions until it gradually replaces the ethanol. Once the tissue is embedded, the xylene is replaced by molten paraffin wax. During these stages, the tissue may shrink because xylene also removes fat residue left in the tissue samples.
Step 6: The Tissue Block
The final stage is called blocking out or infiltration. During infiltration, the last xylene is replaced with molten wax which infiltrates the tissue. Three different wax immersions are used to make sure no clearing agent is left in the tissue sample. After the last infiltration, tissue cassettes removed from the cassettes and placed into a metal mold which is filled with molten wax. The mold is transferred to the cold plate and allowed to set. Once the tissue is set it is ready for the journey of sectioning, making slides, and immunohistochemistry.
In Conclusion
There are many steps necessary to properly prepare tissue samples for histology. The steps must be carried out in a specific order with no deviations to ensure consistency among tissue samples.
References
https://webpath.med.utah.edu/HISTHTML/HISTOTCH/HISTOTCH.html
https://www.leicabiosystems.com/knowledge-pathway/an-introduction-to-specimen-processing/
https://bitesizebio.com/13398/how-histology-slides-are-prepared/
https://www.researchgate.net/publication/233844919_Routine_Histotechniques_Staining_and_Notes_on_Immunohistochemistry
