Tissue processing involves treating tissues to impregnate them with a solid material like paraffin wax to facilitate microscopic sectioning. Proper identification, fixation, and any special post-fixation treatment is important. The goal of tissue processing is to embed tissues in a solid media for support, rigidity for sectioning, and minimal damage. Key steps are completion of fixation, dehydration, clearing, and embedding. Factors like agitation, heat, viscosity, ultrasonic, and vacuum can affect the rate of fluid penetration and impregnation during processing.

1. Tissue processing
HISTOTECHNIQUES
ABDUL QUDDUS (BS-MLT)

2.  Tissue processing refers to any treatment of tissues necessary to
impregnate them with a solid material to facilitate the production of
sections for microscopy.
 Impregnating media include paraffin wax as the most popular media

3. Do’s before processing
 Histopathology tissues are identified by means of a number
 Well organized system of tissue labelling ensures that there is little danger
of incorrect reporting due to error or exchange of tissue identity
 Tissue fixation should be checked if not proper post fixation can be done

4. Special treatment after fixation
 Tissues may need some treatment after the use of some fixatives
 Potassium dichromate fixatives; tissue require thorough washing
in running tap water to remove traces of dichromate
 An insoluble green brown pigment is produced due to reaction with
dehydrating alcohols
 Picric acid fixatives; tissue should not be in contact with water or
aqueous solution prior to dehydration
 Some protein picrate complexes are slightly water soluble and may cause
artefacts unless directly transferred to 80% alcohol

5.  Carnoy’s fliud; tissue should be transferred to 95% or absolute alcohol
 Tissue is already partially dehydrated due to the alcoholic content of the
fixative

6. Principle of tissue processing
 Aim of tissue processing is to
1. Embed the tissue in a solid media to support the tissue
2. Provide sufficient rigidity to the tissue to enable thin sections to be cut
3. Ensure that the media thoroughly solidifies the tissue with little damage

7.  Before embedding the tissue should be subjected to the following steps
1. Completion of fixation
2. Gentle but complete dehydration, to remove aqueous fixative or water
3. Clearing with a substance which is totally miscible with the dehydrating
agent and embedding agent
4. Embedding

8. Factors affecting rate of impregnation
 Agitation; the surest way to impede the interchange of fluids is
 To allow the tissue to sink to the base of container
 Fluid then only disperse through the upper surface and sides
 Agitation allows the removal of contaminated fluid by fresh fluid
 Tissue processing machines are fitted with mechanical agitators
 Too slow agitation is ineffective
 Too fast may cause tissue damage
 Effective agitation reduces processing time by 25-30%

9.  Heat; increases the rate of penetration
 Chilling decreases the rate of penetration
 But overheating the tissue causes shrinkage, brittleness and difficulty in
sectioning
 Also many fluids used are inflammable and heating may cause fire hazard

10.  Viscosity; it affects the rate at which fluid can penetrate the tissue
 The larger the molecule the higher the viscosity and slower the penetration
 Heat may reduce the viscosity in case of wax impregnating process
 But in case of celloidin low viscosity nitrocellulose and resins causes
premature hardening of impregnating media

11.  Ultrasonic; use of ultrasonic is suggested but not widely used
 Vacuum; vacuum is used in every stage of processing
 The only purpose is to remove any air bubbles trapped within the tissue

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