Description
This comprehensive academic note on Tissue Processing provides essential procedural knowledge for tertiary students in Histotechnology, Pathology, and Laboratory Medicine. Derived from the authoritative text "Bancroft's Theory & Practice of Histological Techniques" by Dr. S.K. Suvarna et al., the document meticulously details the sequential steps required to transform fixed tissue into a paraffin-embedded block suitable for microtomy. It explains the principle of replacing tissue water with a supportive medium and outlines the key stages: fixation, dehydration using graded alcohols, clearing with agents like xylene, infiltration with molten paraffin wax, and final embedding. The note covers critical factors influencing processing rates, such as agitation, heat, viscosity, and vacuum, and provides practical guidance on tissue labeling, orientation for optimal sectioning, and selecting appropriate moulds. It compares manual and automated processing methods, highlighting the advantages of modern processors that incorporate temperature control and vacuum cycles. A troubleshooting table addresses common issues like soft tissue, microchattering, and staining irregularities, linking them to specific causes such as inadequate dehydration or reagent saturation. This material is vital for diploma and degree students to master the techniques that ensure high-quality tissue sections for accurate pathological diagnosis. Download this guide to understand the complete workflow and troubleshooting strategies in a histopathology laboratory.
What's inside
Tissue Processing Lecture Objectives At the end of this lecture you must be able to: • Describe tissue processing • Explain stages of tissue processing • Discuss automatic tissue processing • Discuss troubleshooting in tissue processing Introduction •Tissue processing describes the steps required to take tissues from fixation to the state where it is completely infiltrated (permeated) with a suitable wax i.e. paraffin wax to enable cutting of thin sections. Principle of Tissue Processing • Tissue processing is designed to remove all extractable water from the tissue, replacing it with a support medium that provides sufficient rigidity to enable sectioning of the tissue without parenchymal damage or distortion. Factors Influencing the Rate of Processing • When tissue is immersed in fluid, fluid exchange occurs between the fluid within the tissue and the surrounding fluid. • Several factors influence the rate at which the interchange occurs: namely, • Agitation: Increases the flow of fresh solutions around the tissue. Efficient agitation may reduce the overall processing time by up to 30%. • Heat: Increases the rate of penetration and fluid exchange. Care must be taken to avoid shrinkage and hardening of the tissue sample. Factors Influencing the Rate of Processing • Viscosity: Viscosity is the property of resistance to the flow of a fluid. The smaller the size of the molecules in the solution, the faster the rate of fluid penetration (low viscosity).Conversely, if the molecule size is larger, the rate of exchange is slower (high viscosity). • Vacuum: Using pressure to increase the rate of infiltration decreases the time necessary to complete each step in the processing of tissue samples. Vacuum will remove reagents from the tissue, but only if they are more volatile than the reagent being replaced. Vacuum can also aid in the removal of trapped air in porous tissue. Labeling of Tissues • It is important that all specimens are properly labeled before processing is started. • The identification number given to each specimen should be labeled on tissue cassettes which hold specimens when being processed. • For labeling, pen containing ordinary ink should not be used. Printed, or graphite pencil, are satisfactory. Stages of Tissue Processing i. Fixation ii. Dehydration iii. Clearing iv. Infiltration (Impregnation) v. Embedding Fixation • Preserves cells and tissue components with minimal distortion • The most commonly used reagent for the fixation of histological specimens is 10% neutral buffered formalin (NBF). Dehydration • The removal of ‘free’ unbound water and aqueous fixatives from the tissue components. • Dehydrants: Ethanol, Methanol, Butanol, Isopropanol, Acetone, industrial methylated spirit (denatured alcohol). • The most commonly used dehydrant is ethanol. It ensures total dehydration, making it the reagent of choice for the processing of electron microscopy specimens. • Additives to dehydrants: phenol(4%), for softening hard tissue e.g. tendon, nail, dense fibrous tissue etc. Dehydration Ethanol • Fast-acting and reliable. • Graded concentrations of ethanol are used for dehydration; the tissue is immersed in 70% ethanol in water, followed by 95% and 100% solutions. • Should be accomplished slowly. If the concentration gradient is excessive, diffusion currents across the cell membranes may increase the possibility of cell distortion. For this reason, specimens are processed through a graded series of reagents of increasing concentration. • For delicate tissue it is recommended that the processing starts in 30% ethanol. Dehydration • Excessive dehydration may cause the tissue to become hard and shrunken. • Incomplete dehydration will impair the penetration of the clearing reagents into the tissue, leaving the specimen soft and non receptive to infiltration.
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