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Tissue Processing - Histopathology/Histochemistry series

Tissue Processing- Part of Histopathology Series:-

Pre-processing Step: Fixation

Ø The optimal time for fixation will vary between fixatives, tissue type and size.

Ø Dense or fatty tissues usually require more time to be fully fixed.

Ø Most frequently the routine fixative will be neutral buffered formalin.

Pre-processing: Decalcification

Ø Bone and other calcified specimens must be decalcified prior to processing and paraffin infiltration.

Ø Once the mineral has been removed, a standard processing schedule can be used.

Ø Most of the acid decalcifier should be washed away before processing to avoid contaminating the processing reagents.

Ø Note:- Despite complete decalcification, bone (particularly compact bone), will contain dense areas that require thorough processing. In such case choosing a longer schedule is advised.

Pre-processing: Grossing-

Ø For optimal fixation and subsequent high-quality processing the dimensions of tissue specimens are important. Ideally, the thickness should not exceed 4mm and the specimen should fit into a histology cassette without distortion.

Pre-processing: Enclosing-

Ø Cassettes hold and protect the specimen while it undergoes processing. Cassettes chosen must be completely resistant to the solvents and heat used in processing.

Ø They must not distort during use so that there is no chance that the specimen will escape into the processing reagents.

Ø Perforated cassette surfaces must allow for the adequate fluid exchange and proper drainage.

This is a very crucial step and quality of staining is depending upon this step.

Tissue processing is a procedure of removing water from cells and replacing it with a medium which solidifies allowing thin sections to be cut on a microtome. Once the tissue is properly fixed it goes through a process which involves the following steps:

1 Dehydration

2 Clearing

3 Infiltration

Tissue processing is routinely done on an instrument called Tissue processor.

Flowchart of Tissue Processing:-

Tissue processing” describes the steps required to take an animal or human tissue from fixation to the state of complete infiltration with histological paraffin. Subsequently, the processed tissue is made into a paraffin block so it can be sectioned on the microtome.

Step 1: Dehydration -

Ø Alcohol progressively replaces water in all the cells of the specimen.

Ø A series of increasing (typically from 70% to 100%) alcohol concentrations are used to avoid excessive distortion of the tissue.

Ø This is pivotal step, as in third step paraffin embedding is done. Since paraffin is hydrophobic (immiscible i.e. not mixable with water), water inside a specimen must be removed before it can be infiltrated with paraffin. This process is carried out by immersing specimens in a series of alcohol.

Step 2: Clearing-

Ø This solvent displaces the alcohol in the tissue through the process called “clearing”. “Clearing” relates to how clearing agents impart an optical clarity or transparency to the tissue due to their relatively high refractive index.

Ø Another important role of the clearing agent is to remove a substantial amount of fat from the tissue which otherwise presents a barrier to paraffin infiltration.

Ø To make sure that all traces of alcohols are removed from tissues being processed, multiple changes of fresh xylene, clear of carried-over alcohol, are required.

Step 3: Infiltration-

Ø The specimen can now be infiltrated with paraffin. Molten paraffin infiltrates tissues and when cooled solidifies to a consistency that allows sectioning on a microtome.

Ø The amount of structural support given by solidified paraffin can be regulated by choosing different paraffin formulations.

Ø Multiple changes of histological paraffin are required to completely displace the clearing agent.

Ø Paraffin infiltration is greatly enhanced by vacuum.

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