Tissue Processing


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HISTOLOGY LECTURE # 14 INTRODUCTION TO TISSUE PROCESSING Rationale : Processing is a secondary step in the histology process. Tissue after fixation will go through several steps that will allow some structural format to the tissue that will allow the tissue to be sectioned and stain for diagnostic purposes. Objective: Once completed this lecture, the student should be able to: a) Describe the different dehydrants, clearing and infiltrating agents b) Learn the difference between processing options c) Describe the proper and effective way of embedding tissues d) Learn the terms open processor and closed processor. TISSUE PROCESSING INTRODUCTION (Carson Book Pages 26 – 35) All tissues must be adequately supported before they can be sectioned for microscopical examination. The exceptions to this are frozen sections which are sectioned following a range of preparatory freezing methods. Permanent tissues are more commonly taken through a series of reagents and finally infiltrated and embedded in a stable medium which when hard, provides the necessary support for microtomy. This treatment is termed tissue processing. Methods have evolved for a range of embedding media and applications. The most widely used method for routine preparation; sectioning, staining and subsequent storage of large numbers of tissue samples is the paraffin wax method, which we will discuss in the embedding lecture. 1
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2 Principles of tissue processing Tissue processing is concerned with the diffusion of various substances into and out of stabilize porous tissues. The diffusion process results from the thermodynamic tendency of processing reagents to equalize concentrations inside and outside blocks of tissue, thus generally conforming to Fick's Law: the rate of solution diffusion through tissues is proportional to the concentration gradient (the difference between the concentrations of the fluids inside and outside the tissue) as a multiple of temperature dependant constants for specific substances. From this it can be seen that the significant variables in tissue processing are the operating conditions, particularly temperature, the characteristics and concentrations of the reagents and the properties of the tissue. Dehydration (Removal of Water) The first step in processing is dehydration. Water is present in tissues in free and bound (molecular) forms. Tissues are processed to the embedding medium by removing some or all of the free water. During this procedure various cellular components are dissolved by dehydrating fluids. For example, certain lipids are extracted by anhydrous alcohols, and water soluble proteins are dissolved in the lower aqueous alcohols. Dehydration is effected as follows: Dilution dehydration, the most commonly used method. Specimens are transferred through increasing concentrations of hydrophilic or water miscible fluids which dilute and eventually replace free water in the tissues. Chemical dehydration, where the dehydrant, acidified dimethoxypropane or diethoxypropane, is
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