Description
This comprehensive guide on frozen and related sectioning techniques is an essential resource for advanced diploma and degree students in Histotechnology, Pathology, and Laboratory Science. Authored by Dr. S.K. Suvarna and based on the seminal work "Bancroft's Theory & Practice of Histological Techniques," the document provides a detailed examination of the principles, applications, and technical considerations of frozen section preparation. It covers the theoretical basis of rapid tissue freezing, the operation of cryostats, and the management of factors like temperature and ice crystal artifact to ensure diagnostic quality. The material compares the advantages and disadvantages of frozen sections against paraffin-embedded methods, highlighting critical clinical and research uses such as intra-operative diagnosis, enzyme histochemistry, and immunofluorescent techniques. It also delves into specialized procedures like freeze drying and freeze substitution, explaining their stages and research applications. This academic note is designed to support tertiary students and laboratory professionals in mastering these complex techniques for accurate tissue analysis and diagnosis. Review this detailed material to enhance your practical skills in histological preparation.
What's inside
Frozen & Related Sections Frozen Sections • Frozen sections are produced without the use of dehydration steps or overnight fixation. • Needed: OCT (Optimal Cutting Temperature embedding material), mould(chuck) and means of rapidly freezing tissue. • Frozen sections have important clinical and research applications. e.g. in Mohs Micrographic Surgery, in animal research setting Principle of Frozen Sections • The rapid freezing of the tissue sample converts the water into ice. The firm ice within the tissue acts as embedding media to cut the tissue. Theoretical Considerations • The consistency of the frozen block may be altered by varying the temperature of the tissue. – Reducing the temperature will produce a harder block; raising the temperature makes the tissue softer. • The majority of non-fatty unfixed tissues section well at −25°C. The sectioning of fixed tissue requires a block temperature of approximately −10°C or warmer. Theoretical Considerations •There is more water in fixed tissue; consequently, the tissue will have a harder consistency, requiring a higher temperature to obtain the ideal consistency for sectioning. N.B for fatty unfixed tissue section well at a colder temperature or surround the tissue with OCT which helps keep the tissue together when cut. Frozen Sections Advantages Disadvantages Quick diagnostic procedure (≈10mins) Difficult to cut serial sections e.g. fatty tissue, bone and delicate structures i.e. ovarian epithelium Every type of staining can be done Sections cut are thicker. Minimal shrinkage of tissues as compared to paraffin sections Storage can be expensive (use of specialized equipment e.g. ultra-low temperature freezer) Lipids and enzymes which are lost in routine paraffin sections can be demonstrated Formation of ice-crystals can cause distortion in tissue morphology & difficult sectioning Uses of Frozen Sections 1) Rapid production of sections for intra-operative diagnosis. 2) Diagnostic and research enzyme histochemistry for labile enzymes. 3) Diagnostic and research non-enzyme histochemistry, e.g. lipids and some carbohydrates. 4) Immunofluorescent methodology. 5) Immunohistochemistry techniques when heat and fixation may inactivate or destroy the antigens. Freezing of Fresh Unfixed Tissue • The specimen should be frozen as rapidly as is possible without creating freeze artifacts. • Examples of techniques for suitable freezing include: i. Liquefied nitrogen (−190°C) ii. Isopentane (2-methylbutane) cooled by liquid nitrogen (−150°C) iii. Dry ice (−70°C) Ice crystal artifact in a section of skeletal muscle • When freezing tissue for frozen sections, freeze artifact may occur. • The water in the tissue freezes and forms ice crystals, and the crystal size and the quantity of crystals is proportional to the speed at which the tissue is frozen. • The tissue is cut and placed on a microscope slide at room temperature; at this point the tissue is thawed. The thawing of the ice crystals produces freeze artifact that appears as holes or discontinuation of the tissue architecture when viewed microscopically. Fixed Tissue and the Cryostat • The effect of freezing unfixed tissue is the diffusion of labile substances. – Diffusion of labile substances is enhanced when the section is cut in the cryostat, producing heat which causes slight thawing of the cut section. – This may not cause a problem for diagnosis, but it can affect the accurate localization of some abundant enzymes, such as acid and alkaline phosphatases. • The tissue must be fresh and placed in formal calcium at 4°C for 18 hours. The Cryostat • Is a refrigerated cabinet in which a specialty microtome is housed. 1. Smooth turning handwheel with 360∘ locking mechanism. 2. Double compressor system allows for fast freezing and robust lifespan of cryostat 3. Waste collection for liquid condensate is safe and easily accessible The Cryostat
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