Those who are unfamiliar with histotechnology often ask, “How does a pathologist look at a biopsy under the microscope and diagnose a disease?” The process is long and involved, and each sequential step is completely dependent on the previous one. The majority of surgical tissue is treated with various reagents used to preserve tissue elements (such as nuclei, cytoplasm, and tissue morphology) and prepare the specimen for paraffin embedding and microtomy.
Although there are other tissue preparation techniques, such as freezing fresh tissue or embedding tissue in plastic for electron microscopy, the focus of this course is strictly paraffin processing of formalin-fixed tissue samples. Preparing fresh tissue samples for microscopic use has many steps, such as fixation and processing, embedding and microtomy, deparaffinization and staining, and finally coverslipping. This course summarizes the most common processing steps utilized in histology today.
Conventional tissue processing proceeds in the following reagent order. Completion of each step is dependent on the previous step.
| Step 1 | Fixation | - Stabilizes tissue proteins to prevent further changes, such as decay
- Typically the first step on the automated processor
- Most commonly achieved with 10% neutral buffered formalin (NBF)
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| Step 2 | Dehydration | - Removes water from tissue
- Gradual removal of water from tissue is preferred through the use of graded alcohols, typically beginning with 70% and finishing with several changes of 100% alcohol
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| Step 3 | Clearing | - Removes the dehydrant from tissue in preparation for infiltration; begins process of making tissues firm.
- Can be accomplished with xylene, xylene substitutes or isopropyl alcohol
- A minimum of 2 changes of clearant are needed.
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| Step 4 | Paraffin infiltration | |
In the next section, let us consider each tissue processing step and how the reagents affect the specimens.
