Published online: 2019-08-29 THIEME Review Article 51 Fixation and Fixatives: Roles and Functions—A Short Review Himanshu Singh1 Kundendu Arya Bishen1 Deepti Garg2 Hemani Sukhija1 Dheeraj Sharma1 1 Urvashi Tomar 1Department of Oral and Maxillofacial Pathology, Index Institute of Address for correspondence Kundendu Arya Bishen, MDS, PhD, Dental Sciences, Malwanchal University, Indore, Madhya Pradesh, India Department of Oral and Maxillofacial Pathology, Index Institute 2Department of Oral and Maxillofacial Pathology, Bhojia Dental of Dental Sciences, Malwanchal University,Indore 452016, College, Baddi, Himachal Pradesh, India Madhya Pradesh, India (e-mail: [email protected]). Dent J Adv Stud 2019;7:51–55 Abstract Fixation is considered as physiochemical process where cells or tissues are fixed chem- Keywords ically. Fixatives perform various functions such as prevention of autolysis and tissue ► fixatives putrefaction. Various fixative agents include formaldehyde, glutaraldehyde, osmium ► fixation tetroxide, glyoxal, picric acid, and so on. A detailed search on PubMed, Google scholar, ► formaldehyde and Scopus database showed very few articles on “fixation” and “fixative.” Keeping this ► glutaraldehyde fact in mind, a comprehensive review on fixation and fixatives was prepared. The main ► glyoxal aim of this review is to make pathologists and laboratory technicians familiar with the ► osmium tetroxide basic aspects and different types of fixatives. ► picric acid Introduction as putrefaction (bacterial attack) of tissues. Autolysis seems to be a frequent issue in enzyme-rich tissues, and rigorously Fixation is known as a physiochemical process in which cells autolyzed tissue does not get stained properly for microscop- or tissues are fixed chemically. As a result, the tissue or cell ic examination. On the other hand, bacterial invasion can be can combat the successive treatment by different reagents blocked by following the strict antiseptic methods. 1 with negligible disfigurement of morphology. Another important function is conserving the association An ideal fixation involves complicated progression of in between cells and extracellular substances. Fixatives sta- chemical episodes. An ideal fixative is presumed to transmit bilize the cell component by making them insoluble, there- mechanical toughness to tissue so that it resists destruction by reducing the alteration by subsequent treatment and due to further processing steps. It prevents the autolysis, also preventing osmotic damage of tissue, which may cause putrefaction of tissue as well as tissue component degrada- shrinkage or swelling, thus preserving the cellular and tissue 1,2 tion. Fixation should be able to preserve the cellular struc- structure in life-like state. 2 ture and tissue architecture in life-like manner. Fixation also performs various other functions such as For the purpose of tissue processing in the histopatholo- making tissue firm, so that gross cutting becomes much easi- gy, fixation of tissue is considered as necessary and essential er. Also, fixatives help make the tissue more easily permeable step. Fixation amends the physio-chemical state of tissues so for subsequent reagents and play a role in emphasizing the that it remodels the reactiveness of cellular components for dissimilarity in refractive index and thus help in increased 3 stains. Fixatives can be classified in different ways, as shown visibility of different elements of tissue.1,2,4 in ►Tables 1 –3. Factors Affecting Fixation and Fixatives Functions of Fixative Length of Fixation Fixatives perform various functions. The primary function The ideal time of fixation is experimentally determined for of fixatives is to prevent autolysis (enzymes attack) as well different types of tissue. If time period for fixation is longer, received DOI https://doi.org/ ©2019 Bhojia Dental College and April 6, 2019 10.1055/s-0039-1693098 Hospital affiliated to Himachal accepted ISSN 2321-1482. Pradesh University May 20, 2019 published online August 29, 2019 52 Fixation and Fixatives Singh et al. Table 1 Classification of fixatives based on chemical Size composition Tissue thickness is one of the important factors for fixation. If the sample size is large, it is unfavorable for the fixative Fixatives Examples to penetrate and reach to the deeper part of the tissue, 1. Physical agents Heat, microwaves which would result in autolysis of epithelium.4 Ideally 4- to 2. Aldehydes Formaldehyde, acrolein, 6-mm-thick specimen is best suited for complete penetration glutaraldehyde by fixatives. 3. Coagulants Methyl alcohol, ethyl alcohol, acetic acid Osmolarity 4. Oxidizing agents Osmium tetroxide If osmolarity of tissue as well as fixative is same, it will pre- 5. Miscellaneous Picric acid, mercuric chloride vent swelling or shrinkage of the tissue.7 Table 2 Classification of fixatives based on number of struc- Various Fixating Agents Used in tures fixed Histopathology Fixatives Examples Formaldehyde or Formalin 1. Simple fixatives e.g., Formaldehyde, picric acid, Formaldehyde was discovered in 1859 by Butlerov. In 1889 osmium tetroxide Ttrillat was the first who manufactured formaldehyde com- 2. Compound fixatives e.g., Bouin’s fluid, formol saline, mercially as industrial reagent. In 1892, Ferdinand Blum recog- Zenker’s fluid nized that formalin could give benefit when used as a fixative.8,9 The most routinely used solution for fixation of tis- Table 3 Classification of fixatives based on type of structures sue—10% formalin solution v/v—is nothing but an aqueous fixed suspension of formaldehyde In 10% neutral buffered form, formaldehyde is found to be the most commonly used fix- Fixatives Examples ative in pathology. Reaction between the formaldehyde 1. Histochemical Formaldehyde, glutaraldehyde, vapor and macromolecules of tissue seems to be complex. Form- fixatives fixatives aldehyde reacts with nucleic acids as well as proteins, and 2. Microanatom- Bouin’s fluid, 10% formalin, Zenker’s it penetrates between nucleic acids and proteins and forms ical fixatives fluid, formol calcium, Heidenhain’s stabilized shell of nucleic acid-protein complex.10-13 As com- susa, Helly’s fluid, Rossman’s fluid, pared with other fixatives, formaldehyde causes lesser tissue 3. Cytologic Champy’s fluid, glacial acetic acid, shrinkage, with exceptions being acetone and ethanol. Form- fixatives alcohol, formol saline, Carnoy’s fluid, Clarke’s fluid, Newcomer’s fluid, aldehyde seems to harden tissue more when compared with Flemming’s fluid other fixatives. The lipids are conserved, but carbohydrates are not fixed by formaldehyde.4 it results in over–cross-linking, and samples become brittle. Formalin comprises 37 to 40% formaldehyde and 60 to If time period for fixation is short, sufficient amount of pen- 63% water by weight. After continuous storage for long peri- etration in tissues and cross-linking will not occur.4 For oral ods, accumulations of white deposits are observed in the soft tissue, overnight fixation is sufficient. solution. These are the precipitates of paraformaldehyde. By storing formalin at low temperature, these white deposits Temperature can be avoided. Also, 10% methanol may be added into the Temperature of fixative during fixation may affect the tis- formalin to minimize the polymerization reaction that pro- sue architecture. Rate of fixation is increased with increase duces paraformaldehyde precipitate. It also contains a slight in temperature, but increased temperature will also increase amount of formate ions. These are obtained from Cannizza- autolysis rate. If temperature is low or decreased, the dif- ro reaction. In this reaction, two molecules of formaldehyde fusion rate also decreases, which results in extended pen- react together. One molecule condenses to form methanol 14 etration time.5 For electron microscopic studies, 0°to 4°C is and second molecule gets oxidized to form formic acid. The appraise as ideal temperature. solution is acidic in reaction because of formic acid, but acid- ic nature of solution can be counterbalanced with incorpora- 15 Concentration tion of magnesium carbonate in little proportion. Fixative agents need prolonged time for fixation if concen- tration is low. If concentrations of fixing agent are high, it Glutaraldehyde results in damaging of cellular structures as well as obliterat- Glutaraldehyde was found in 1963 by Sabatini et al as par- ed enzyme activities.6 Different fixatives have different ideal ticular fixative for ultrastructural researches. Glutaraldehyde concentration that is determined experimentally; for exam- comprises two aldehyde groups that are divided by three ple, ideal fixative for oral soft tissue is formalin used in 10% methylene bridges. Although penetration rate of glutaralde- concentrated solution. hyde is found to be slower when compared with formaldehyde, Dental Journal of Advance Studies Vol. 07 No. 2/2019 Fixation and Fixatives Singh et al. 53 glutaraldehyde is more effective cross-linker for proteins than Glyoxal formaldehyde and it also inhibits enzyme activity.16-18 Glyoxal was first advocated in 1943. They are also known When polymerization of aqueous solution of glutaralde- as ethanedial or oxalaldehyde. Glyoxal is considered as hyde occurs, it forms oligomeric and cyclic compounds, and alternative fixative to formalin because it is a dialdehyde also forms glutaric acid by oxidation. For stability, it requires in nature. It is a bifunctional aldehyde. Its individual alde- pH of 5 and storage at 4°C.19,20 At room temperature, glutaral-