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The page below is a sample from the LabCE course Tissue Processing in Histology. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Processing Steps Defined

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.

OrderProcessing StepPurpose/Comments
Step 1Fixation
  • 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)
Step 2Dehydration
  • 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
Step 3Clearing
  • 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.
Step 4Paraffin infiltration
  • Displaces clearing agent in tissue with paraffin, in preparation for paraffin embedding
  • Three or four changes of paraffin are necessary for complete impregnation
  • Paraffin creates stability in the tissue by hardening/making firm so that it may be sectioned during microtomy
  • Paraffin assists in preparing tissue for embedding

In the next section, let us consider each tissue processing step and how the reagents affect the specimens.