Description
This comprehensive academic note on Tissue Fixation provides a detailed foundation 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 thoroughly explains the chemical process of fixation, its principle of crosslinking and coagulating proteins to preserve tissue architecture. It outlines the key aims, including preventing autolysis, hardening tissues for manipulation, and enhancing staining properties. The note details the ideal properties of a fixative and extensively covers the critical factors affecting fixation, such as volume, duration, temperature, pH, and tissue type. It classifies fixatives by their mechanism of action and primary use, providing examples like aldehydes, oxidizing agents, alcohols, mercurials, and picrates, along with their specific applications, advantages, and disadvantages. Tables listing the composition and recommended use for various fixatives, such as 10% Formalin, Zenker's fluid, and Bouin's solution, offer practical guidance. This material is essential for diploma and degree students to understand the selection, application, and limitations of different fixatives to ensure optimal tissue preservation for accurate microscopic diagnosis. Download this guide to master the principles and practices of tissue fixation in a histopathology laboratory.
What's inside
Tissue Fixation Lecture Objectives At the end of this lecture, you must be able to: • Define fixation and state its principle • Discuss aims of fixation • State ideal properties of fixatives • Discuss factors that affect fixation • Describe methods of tissue fixation • Know the classification of fixatives and give examples • State advantages and disadvantages of various fixatives Introduction • Fixation is a chemical process by which biological tissues are preserved from decay by exposing the tissue to chemical compounds, called fixatives. • Principle of fixation The fixative brings about crosslinking, denaturation or coagulation of proteins so that the semifluid state is converted into semisolid state; so that it maintains everything in vivo in relation to each other. Thus semisolid state facilitate easy manipulation of tissue. Introduction Aims of Fixation • To preserve the tissues as close to their living state as possible • To prevent autolysis and bacterial attack – Fixatives are toxic to microorganisms or bacteria which may be present or attack the tissue. They also disable intrinsic biomolecules e.g. proteolytic enzymes which may digest the cell. • To harden the tissues – The hardening effect of fixatives allows easy manipulation of soft tissue like brain, intestines etc.) • To prevent tissues from changing their shape and size during processing Aims of Fixation • To allow clear staining of sections subsequently – Certain fixatives like formaldehyde intensifies the staining character of tissue especially with haematoxylin • To improve the optical differentiation of cells & tissues – Fixatives alter to varying degrees the refractive indices of the various components of cells and tissues so that stained components are more easily visualized than when unfixed General Properties of fixatives 1. Coagulation and precipitation. 2. Penetration – Fixation is done by immersing the tissue in fluid containing the fixative. The faster a fixative can penetrate the tissue the better it is. penetration power depends upon the molecular weight e.g. formalin fixes faster than osmic acid 3. Solubility – All fixatives should be soluble in a suitable solvent, preferably in water so that adequate concentrations can be prepared General Properties of fixatives 4. Concentration – It is important that the concentration of fixative is isotonic. Excessively hypertonic or hypotonic solutions can lead to cell membrane damage 5. Reaction – Most fixatives are acidic. It may help in fixation but can affect staining so has to be neutralized e.g. formalin is neutralized by adding of calcium carbonate. [breakdown of formaldehyde to form formic acid produces an acidic solution which in turn reacts with hemoglobin to produces an artefact pigment (acid formaldehyde hematin] Properties of an ideal fixative • Prevents postmortem changes. • Preserves tissue in their natural state and fix all components. • Make the cellular components insoluble to reagent used in tissue processing. • Preserves tissue volume. • Avoid excessive hardness of tissue. • Allows enhanced staining of tissue. • Should be non toxic and non allergic for user • Not very expensive. • Allow cell parts to become clear visible by means of dyes and improved refractive indices . Properties of an ideal fixative • Should be readily disposable or recyclable • Have a shelf life of at least one year • Be compatible with modern automated tissue processors. • Being useful for a wide variety of tissues. • Support specialized procedures such as histochemical, immunohistochemical, in situ hybridization, etc. • Should permit the recovery of macromolecules including proteins, mRNA, and DNA without extensive biochemical modifications from fixed and paraffin embedded tissues Factors Affecting Fixation • Volume of the Fixative • Duration of fixation • Time interval from removal of tissues to fixation • The rate of penetration of the fixing fluid • Choice of fixative • Nature of tissue • Size and thickness of piece of tissue • Agitation • pH • Osmolarity • Temperature Factors Affecting Fixation • Volume of the Fixative – It is important to have an excess volume of fixative in relation to the total volume of tissue because with additive fixatives (cross linking fixatives) the effective concentration of reagent is depleted as fixation proceeds and in a small total volume this could have an effect on fixation quality. At least 10-20 …
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