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This chapter discusses the basics of fixation, along- side the advantages and disadvantages of specific fixatives. It also provides some of the formulas for these fixatives currently used in pathology, histol- ogy and anatomy.
It is fair to say that the appropriate fixation of tissues for histological examination is central to all histology tests, as without this process all tissues would degrade and analysis would be useless. The last century has seen the development of a range of fixatives, with few recent modifications. The mecha- nisms and principles by which specific fixatives act fall into several broad groups. These include the covalent addition of reactive groups and cross-links, dehydration, the effects of acids, salt formation, and heat. Compound fixatives may function using sev- eral of these mechanisms.
When choosing a fixative there is a balance between the advantages and disadvantages which each fixative possesses. These include molecular changes or losses from ‘fixed’ tissues, swelling or shrinkage of tissues, variations in the quality of histochemical and immunohistochemical staining, the effect on biochemical analysis and the ability to maintain the structure of cellular organelles.
The major objective of fixation in pathology is to maintain clear and consistent morphological fea- tures (Eltoum et al., 2001a, 2001b; Grizzle et al., 2001). The development of specific fixatives has usually been empirical, although much of the understand- ing of the mechanisms of fixation has been based upon information obtained from leather tanning and vaccine production. In order to visualize the 40
microanatomy of stained tissue sections, the origi- nal microscopic relationships between cells, cel- lular components (e.g. the cytoplasm and nuclei) and the extracellular material must be maintained with little disruption to the organization of the tis- sue. The local chemical composition of the tissue must also be maintained. Many tissue components are soluble in aqueous acid or other liquid environ- ments and to reliably view the microanatomy and microenvironment of these tissues the soluble com- ponents must not be lost during fixation and tissue processing. Minimizing the loss of cellular compo- nents which include large proteins, small peptides, mRNA, DNA and lipids, prevents the destruction of macromolecular structures such as cytoplasmic membranes, smooth endoplasmic reticulum, rough endoplasmic reticulum, nuclear membranes, lyso- somes and mitochondria. Each fixative, combined with the tissue processing protocol, maintains some molecular and macromolecular aspects of the tissue better than other fixative/processing combinations. If soluble components are lost from the cytoplasm of cells, the color of the cytoplasm on hematoxylin and eosin (H&E) staining will be reduced or modified and aspects of the appearance of the microanatomy of the tissue, e.g. mitochondria, will be lost or dam- aged. Similarly, immunohistochemical evaluations of structure and function may be reduced or lost.
Almost any method of fixation induces shrinkage or swelling, hardening of tissues and color variations in various histochemical stains (Sheehan & Hrapchak, 1980; Horobin, 1982; Fox et al., 1985; Carson, 1990; Kiernan, 1999; O’Leary & Mason, 2004). Various methods of fixation always produce some artifacts in the appearance of tissue on staining. However, for

Types of fixation
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diagnostic pathology it is important that such arti- facts are consistent, predictable and understood.
The chosen fixative acts by minimizing the loss or enzymatic destruction of cellular and extracellular molecules, maintaining macromolecular structures and protecting tissues from destruction by microor- ganisms. This results in one view of a dynamically changing, viable tissue (Grizzle et al., 2001). The fixative should also prevent the subsequent break- down of the tissue or molecular features by enzy- matic activity and/or microorganisms during long term storage. These tissues removed from patients are an important resource which may at a later stage be subjected to further specialized tests, e.g. DNA- related or gene analysis.
A fixative not only interacts initially with the tissue in its aqueous environment but it also has ongoing reactivity with any unreacted fixative and the chemi- cally altered tissues. Fixation interacts with all phases of processing and staining from dehydration to staining of tissue sections using histochemical, enzymatic or immunohistochemical stains (Eltoum et al., 2001b; Rait et al., 2004). It follows that any stained tissue section, produced after specific fixa- tion combined with tissue processing, is a compro- mise of fixed tissue changes formed from the natural living tissue.
To date, a universal or ideal fixative has not been identified. Fixatives are therefore selected based on their ability to produce a final product needed to demonstrate a specific feature of a specific tissue (Grizzle et al., 2001). In diagnostic pathology, the fixative of choice for most pathologists has been 10% neutral buffered formalin (Grizzle et al., 2001).
An important constraint in using formaldehyde has been the loss of antigen immunorecognition due to this type of fixation combined with processing the tissue to paraffin wax (Eltoum et al., 2001a, 2001b). However, from a clinical perspective the advent of heat-induced epitope retrieval methods, instigated in the early 1990s, have overcome many of these limitations (Shi et al., 1991). Similarly, the analysis of mRNA and DNA from formalin-fixed, paraffin- embedded tissue has been problematic (Grizzle et al., 2001; Jewell et al., 2002; Steg et al., 2006; Lykidis et al., 2007). All widely used fixatives are therefore
selected by compromise, with their positive aspects balancing against their less desirable features.
The most important characteristic of a fixative is to support high quality and consistent staining with H&E, both initially and after storage of the paraffin blocks for at least a decade, although new guidelines within the United Kingdom recommend that paraf- fin processed blocks are now kept for 30 years. The fixative must have the ability to prevent short and long term destruction of the micro-architecture of the tissue by stopping the activity of catabolic enzymes and hence autolysis, minimizing the diffusion of sol- uble molecules from their original locations. Another important characteristic of a good fixative, which helps maintain tissue and cellular integrity, is the fixation and inactivation of infectious agents.
It is also important to have good toxicological and flammability profiles which permit the safe use of the fixative (Grizzle & Fredenburgh, 2005). The advent of new biological methods, increased understanding of the human genome and the need to rapidly evaluate the biology of disease processes means that fixatives should also permit the recov- ery of macromolecules including proteins, mRNA, and DNA from fixed and paraffin-embedded tissues without extensive biochemical modifications.
Other important characteristics of an ideal fixa- tive include being useful for a wide variety of tissue types, including fat, lymphoid and neural tissues. It should preserve small and large specimens and support histochemical, immunohistochemical, in situ hybridization and other specialized procedures. The fixative should penetrate and fix tissues rapidly, have a shelf life of at least one year and be compat- ible with modern automated tissue processors. It should be readily disposable or recyclable, support long term tissue storage to give excellent microt- omy of paraffin blocks and should be cost effective