Fixatives

Fixatives used in histopathology and cytopathology divisions of pathology

1. Fixatives Aseem Jain Resident of Pathology

2. Tissue processing After removal from the body the tissues are exposed to a series of reagents that fix, dehydrate, clear, and infiltrate the tissues. The tissue is finally embedded in a medium that provides support for microtomy. Every step of the tissue processing is important. Preserving cells and tissue components with minimal distortion is the most important aim of processing tissue processing .

3. Fixation This is a process by which constituents of cells and tissue are fixed so that they can withstand subsequent treatment with various reagents with minimum loss of architecture. The reagents by which this process is achieved are known as ‘Fixatives’. The major objective of fixation in pathology is to maintain clear and consistent morphological features. Without attention to this process, the range of tests performed in a modern histopathology laboratory will be rendered ineffective and practically useless.

4. Why to fix.?? To make the cells and tissue capable to handle the subsequent steps in tissue processing. To prevent bacterial degradation of tissue. To ensure good staining of the tissue. To prevent further autolysis of the tissue by inactivating the lysosomal enzymes. To make the tissue useful for various special procedures like IHC etc.

5. A Good Fixative… Supports high quality and consistent staining with H&E. Prevents short- and long-term destruction of the micro-architecture of the tissue. Inanimates infectious agents. Is less toxic and less inflammable. Permits the recovery of macromolecules (proteins,mRNA,DNA) without extensive biochemical modifications. Is useful in a wide variety of tissues. Penetrates & fixes tissues rapidly . Has a shelf life of at least 1 year. Is compatible with modern automated tissue processors. Is cost effective and easily disposable.

6. 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. In diagnostic pathology, the fixative of choice for most pathologists has been 10% neutral buffered formalin.

7. Types of fixation Physical fixation Heat fixation – Simplest form of fixation. Microwave fixation – Speeds fixation ; Reduces time of fixation from >12 hours to < 20 minutes. Freeze drying & Freeze substitution Chemical fixation Utilizes organic or non-organic solutions to maintain adequate morphological preservation. Chemical fixatives – Coagulant, Cross linking, Compound.

8. Types of chemical fixatives

9. Coagulant fixatives Both organic and non-organic solutions may coagulate proteins, making them insoluble. Cellular architecture is maintained primarily by lipoproteins and by fibrous proteins such as collagen; coagulating such proteins maintains tissue histomorphology at the light microscopic level. They result in cytoplasmic flocculation and poor preservation of mitochondria so not useful for electron microscopy.

10. Dehydrating coagulant fixatives Alcohols (Methanol and Ethanol) & Acetone . Methanol is structurally more similar to water than ethanol ; So Fixation begins at a concentration of 50-60% for ethanol but requires a concentration of 80% or more for methanol. Removal and replacement of free water from tissue by any of these agents has several potential effects on proteins within the tissue.

11. Water – involved in hydrophobic as well as hydrophilic bonding of protein. So if tissue is dehydrated i.e. Water is removed, the tertiary structure of proteins is disrupted or the structure becomes partially reversed (hydrophobic groups moving to outer surface of proteins). Disruption of the tertiary structure of proteins, i.e. denaturation, changes their physical properties, potentially causing insolubility and loss of function.

12. Other coagulant fixatives – Acidic coagulants such as picric acid and trichloroacetic acid change the charges on the ionizable side chains of proteins and disrupt electrostatic and hydrogen bonding. They also insert a lipophilic anion into a hydrophilic region and hence disrupt the tertiary structures of proteins. Acetic acid coagulates nucleic acids but does not fix or precipitate proteins; it is therefore added to other fixatives to prevent the loss of nucleic acids.

13. Cross linking fixatives Formaldehyde, glutaraldehyde and other aldehydes e.g. chloral hydrate and glyoxal, metal salts such as mercuric and zinc chloride, and other metallic compounds such as osmium tetroxide. They have actions of forming cross-links within and between proteins and nucleic acids as well as between nucleic acids and proteins. “Covalent additive fixatives”

14. Formaldehyde fixation Formaldehyde in its 10% neutral buffered form (NBF) is the most common fixative used in diagnostic pathology. Formaldehyde is commercially supplied as a 37–40% solution and in the following formulae is referred to as 37% formaldehyde. 10% Neutral Buffered formalin - Tap water = 900 ml Formalin (37% formaldehyde solution) = 100 ml Sodium phosphate, monobasic, monohydrate = 4 g Sodium phosphate, dibasic, anhydrous = 6.5 g pH should be 7.2-7.4.

15. In an aqueous solution formaldehyde forms methylene hydrate, a methylene glycol as the first step in fixation. Methylene hydrate reacts with several side chains of proteins to form reactive hydroxymethyl side groups (–CH2–OH). The formation of hydroxymethyl side chains is probably the primary and characteristic reaction. The formation of actual cross-links may be relatively rare at the currently used short times of fixation.

16. Formaldehyde also reacts with nuclear proteins and nucleic acids. It penetrates between nucleic acids and proteins and stabilizes the nucleic acid-protein shell, and it also modifies nucleotides by reacting with free amino groups, as it does with proteins. The side chains of peptides or proteins that are most reactive with methylene hydrate, and hence have the highest affinity for formaldehyde, include lysine, cysteine, histidine, arginine, tyrosine, and reactive hydroxyl groups of serine and threonine.

17. The reactive groups may combine with hydrogen groups or with each other, forming methylene bridges. If the formalin is washed away, reactive groups may rapidly return to their original states, but any bridging that has already occurred may remain. Washing for 24 hours removes about half of reactive groups, and 4 weeks of washing removes up to 90%. So, in the rapid fixation used in diagnostic pathology, most ‘fixation’ with formaldehyde prior to tissue processing stops with the formation of reactive hydroxymethyl groups.

18. When formaldehyde dissolves in an unbuffered aqueous solution, it forms an acid solution – Acid Formalin(pH 5-5.5) Acid formalin may react more slowly with proteins than NBF because amine groups become charged. Acid formalin also preserves immunorecognition much better than NBF. The disadvantage of using acid formalin for fixation is the formation of a brown-black pigment with degraded hemoglobulin.

19. Other formulae of formaldehyde – Carson’s modified Millonig’s phosphate buffered formalin – this formula is reported to be better for ultrastructural preservation than NBF. Formal (10% formalin), calcium acetate – good fixative for preservation of lipids. Formal (10% formalin), saline Formal ( 10% formalin), zinc , unbuffered- is an excellent fixative for immunohistochemistry. Formalin , buffered saline Formalin , buffered Zinc

20. Glutaraldehyde fixation Glutaraldehyde is a bifunctional aldehyde that probably combines with the same reactive groups as does formaldehyde. In aqueous solutions glutaraldehyde polymerizes, forming cyclic and oligomeric compounds and it is also oxidized to glutaric acid. To aid in stability, it requires storage at 4°C and at a pH of around 5.

21. Unlike formaldehyde, glutaraldehyde has an aldehyde group on both ends of the molecule. With each reaction of the first group, an unreacted aldehyde group may be introduced into the protein and these aldehyde groups can act to further cross-link the protein. Extensive cross-linking by glutaraldehyde results in better preservation of ultra structure, but this method of fixation negatively affects immunohistochemical methods. Thus, any tissue fixed in glutaraldehyde must be small (0.5 mm maximum) and, unless the aldehyde groups are blocked, increased background staining will result if several histochemical methods are used.

22. Osmium tetroxide fixation Osmium tetroxide (OsO4), a toxic solid, is soluble in water as well as non-polar solvents and can react with hydrophilic and hydrophobic sites including the side chains of proteins, potentially causing cross linking. Osmium tetroxide is known to interact with nucleic acids, specifically with the 2,3-glycol moiety in terminal ribose groups and the 5,6 double bonds of thymine residues. Nuclei fixed in OsO4 and dehydrated with alcohol may show prominent clumping of DNA.

23. Large proportions of proteins and carbohydrates are lost from tissues during osmium fixation; some of this may be due to the superficial limited penetration of OsO4 (i.e. <1 mm) into tissues or its slow rates of reaction. The best characterized reaction of osmium is its reaction with unsaturated bonds within lipids and phospholipids. In addition to its use as a secondary fixative for electron microscope examinations, OsO4 can also be used to stain lipids in frozen sections. Osmium tetroxide fixation causes tissue swelling which is reversed during dehydration steps.

24. Mercuric fixatives Historically, mercuric chloride was greatly favored for its qualities of enhancing the staining properties of tissues, particularly for trichrome stains. Disadvantages of mercuric chloride – Health and safety issues involved with the use of a mercury-containing fixative , Reduced reliance on ‘special stains’, Inevitable formation of deposits of intensely black precipitates of mercuric pigment in the tissues. This subsequently gives them inferior value for immunohistochemical and molecular studies. Mercury-containing chemicals are an environmental disposal problem.

25. Mercuric chloride reacts with ammonium salts, amines, amides, amino acids, and sulfydryl groups, and hardens tissues. It is especially reactive with cysteine, forming a dimercaptide and acidifying the solution. Mercury-based fixatives are toxic and should be handled with care. They should not be allowed to come into contact with metal, and should be dissolved in distilled water to prevent the precipitation of mercury salts. Mercury fixatives are no longer used routinely except by some laboratories for fixing hematopoietic tissues.

26. Other mercuric fixatives – Zenker’s solution – Just before use add 5ml of glacial acetic acid to 95 ml of above solution. This is good fixative for bloody(congested) specimens and trichrome stains. Helly’s solution Schaudinn’s solution Ohlmacher’s solution Carnoy- Lebrun solution- this fixative penetrates rapidly. B5 fixatives- Frequently used for bone marrow , lymph nodes ,spleen , and other hematopoietic tissues.s

27. Fixatives for electron microscopy The preferred fixatives are a strong cross linking fixative such as - Glutaraldehyde, A combination of glutaraldehyde and formaldehyde, or Carson’s modified Millonig’s, followed by post-fixation in an agent that further stabilizes as well as emphasizes membranes such as OsO4.

28. Fixatives for DNA, RNA & Protein analysis HOPE (HEPES- glutamic acid buffer mediated Organic Solvent Protection Effect) fixative. 2. Reversible cross-linker dithio-bis[ succinimidyl propionate] (DSP) for immunocytochemistry and expression profiling, in addition to zinc-based fixatives.

29. Metallic ions as fixative supplement Several metallic ions have been used as aids in fixation, including Hg2+, Pb2+, Co2+, Cu2+, Cd2+, [UO2]2+, [PtCl6]2+, and Zn2+. Mercury, lead, and zinc are used most commonly in current fixatives, e.g. zinc containing formaldehyde is suggested to be a better fixative for immunohistochemistry than formaldehyde alone.

30. Compound fixatives Other agents may be added to formaldehyde to produce specific effects that are not possible with formaldehyde alone. Formaldehyde + Ethanol (dehydrant) = Alcoholic formalin. Alcoholic formalin – This combination preserves molecules such as glycogen and results in less shrinkage and hardening than pure dehydrants. For fixation of some fatty tissues, such as breast, in which preservation of the lipid is not important. Fixation of gross specimens in alcoholic formalin may aid in identifying lymph nodes embedded in fat. Good at preserving antigen immunorecognition, but non-specific staining or background staining in immunohistochemical procedures can be increased.

31. Factors affecting quality of fixation Buffer and pH. Duration of fixation & Size of tissue. Temperature of fixative. Concentration of fixative. Osmolality of fixatives and ionic composition.

32. Buffer and pH In a strongly acidic environment, the primary amine target groups (–NH2) attract hydrogen ions (–NH+3) and become unreactive to the hydrated formaldehyde (methylene hydrate or methylene glycol), and carboxyl groups (–COO−) lose their charges (–COOH). This may affect the structure of proteins. The extent of formation of reactive hydroxymethyl groups and cross-linking is reduced in unbuffered 4% formaldehyde, which is slightly acidic.

33. At the acidic pH of unbuffered formaldehyde, hemoglobin metabolic products are chemically modified to form a brown-black, insoluble, crystalline, birefringent pigment. To avoid the formation of formalin pigment, neutral buffered formalin is used as the preferred formaldehyde-based fixative. Acetic acids and other acids work mainly through lowering pH and disrupting the tertiary structure of proteins. Buffers are used to maintain optimum pH. Commonly used buffers - Phosphate, tris, cacodylate, bicarbonate & acetate

34. Duration of fixation and Size of tissue The depth (d) reached by a fixative is directly proportional to the square root of duration of fixation (t) and expressed this relation as: d = k √t (k=Constant of diffusibility). Thus, for most fixatives, the time of fixation is approximately equal to the square of the distance which the fixative must penetrate. Gross specimens should not rest on the bottom of a container of fixative: they should be separated from the bottom by wadded fixative-soaked paper or cloth, so allowing penetration of fixative or processing fluids from all directions. In addition, unfixed gross specimens which are to be cut and stored in fixative prior to processing should not be thicker than 0.5cm.

35. Proteins inactivate fixatives, especially those in blood or bloody fluids. Bloody gross specimens should therefore be washed with saline prior to being put into fixative. The fixative volume should be at least 10 times the volume of the tissue specimen for optimal, rapid fixation. It has been suggested that rapid fixation is acceptable as long as histochemical staining remains adequate; and that immunohistochemistry and other molecular techniques are probably enhanced by shorter times of fixation using an aldehyde based fixation.

36. Temperature of fixative The diffusion of molecules increases with rising temperature due to their more rapid movement and vibration; i.e. the rate of penetration of a tissue by formaldehyde is faster at higher temperatures. Microwaves therefore have been used to speed formaldehyde fixation by both increasing the temperature and molecular movements.

37. Concentration of fixative Effectiveness and solubility primarily determine the appropriate concentration of fixatives. Concentrations of formalin above 10% tend to cause increased hardening and shrinkage. Ethanol concentrations below 70% do not remove free water from tissues efficiently.

38. Osmolality of fixatives and ionic composition The Osmolality of the buffer and fixative is important; hypertonic and hypotonic solutions lead to shrinkage and swelling, respectively. The best morphological results are obtained with solutions that are slightly hypertonic (400–450 mOsm), though the osmolality for 10% NBF is about 1500 mOsm. Similarly, various ions (Na+, K+, Ca2+, Mg2+) can affect cell shape and structure regardless of the osmotic effect.

39. Fixation artefacts During fixation, tissues commonly change in volume.  Some intercellular structures such as collagen swell when fixed. Tissues fixed in formaldehyde and embedded in paraffin wax shrink by 33%. The nuclei in frozen sections are usually bigger that those of the same tissue which has been subjected to conventional preparation.  Prolonged fixation in formalin can give rise to secondary shrinkage. Hypertonic solutions give rise to cell shrinkage; isotonic and hypotonic fixatives to cellular swelling and poor fixation. 

40. Artefacts related to diffusion of unfixed material : Diffusion of unfixed material may produce false localization due to movement to some place other than its original location. For example, false localization occurring with glycogen is known as streaming artefact False fixation of extraneous material to tissue : This may occur in autoradiography with (H 3 ) labeled amino acids, sugars, thymidine and uridine . Tissues may incorporate these substances into themselves by active metabolism resulting in too high localization of various radioactively labeled substances.

41. Improper Fixation : Delay in fixation or inadequate fixation produces changes like- Altered staining quality of cells Cells appear shrunken and show cytoplasmic clumping Indistinct nuclear chromatin with nucleoli sometimes not seen Vascular structures, nerves and glands exhibit loss of detail Impression of scar formation or loss of cellularity.

42. Use of improper fixative : Fixation in alcohol results in poor staining of the epithelium and improper fixation of the connective tissue. Collagen bundles have an amorphous appearance that is not a result of scar formation but rather result of artefact. Alcohol fixes tissues but causes severe shrinkage. Therefore, alcohol is not recommended as a substitute for formalin except in extreme emergencies. It also makes the tissue brittle, resulting in microtome sectioning artefacts with chattering and a Venetian blind appearance. Currently, 10% neutral buffered formalin is highly recommended for routine fixation purposes. One excellent indicator of poor fixation is the loss of detail of extravasated RBC's.

43. Microtome sectioning artefact

44. Fixation artefact simulating acantholytic disease – Tissue fixed in rehydrated formalin exhibits a prominent acantholysis of superficial epithelium with preservation and attachment of the basal cell layer to the underlying tissue. This acantholytic artefact simulates Pemphigus , Hailey-Hailey disease or Darier's disease. Tissues allowed to air-dry will dehydrate, particularly if placed on an adsorbent surface such as gauze sponge. Such tissue cannot be reconstituted and will show dehydration artefact.

45. Cytological Fixation & Fixatives Rapid fixation of smears is necessary to preserve cytologic detail of cells spread on a glass slide that are to be stained by the Papanicolau method. If smears are allowed to air-dry prior to fixation, marked distortion of the cells occurs. Solution of Ether + 95% ethanol – fixative of choice in past. Subsequently, it has been necessary to abandon this original and excellent fixative because ether presents a fire hazard. Ninety-five percent ethyl alcohol (ethanol) is now employed as a fixative by most laboratories, with excellent results.

46. Smears should remain in the 95% ethyl alcohol fixative for a minimum of 15 minutes prior to staining. However, prolonged fixation of several days or even weeks will not materially alter the appearance of the smear. EQUIVALENT CONCENTRATIONS OF SEVERAL ALCOHOLS FOR PURPOSES OF CELL FIXATION 100% Methanol 95% Ethanol 95% Denatured alcohol 80% Propanol 80% Isopropanol Wet fixation with alcohol is recommended for all nongynecologic material to be stained by the Papanicolau method. For gynecologic material, coating fixatives may be used.

47. Coating fixatives A number of agents on the market today can be sprayed or applied with a dropper to freshly prepared smears, thus eliminating the use of bottles and fixing solutions. Most of these agents have a dual action in that they fix the cells and, when dry, form a thin, protective coating over the smear. These fixatives are particularly helpful if the smears must be mailed to a distant cytology laboratory for evaluation. The method is not recommended for smears prepared from fluids within the laboratory.

48. As in any good method of fixation, the coating fixative should be applied immediately to fresh smears. The distance from which the slides are sprayed with an aerosol fixative affects the quality of the cytologic detail. Danos-Holmquist tested several spray fixatives and found that the distance of 10-12 inches was optimal. Aerosol sprays are not recommended for bloody smears because they cause clumping of erythrocytes.

49. Coating fixatives may also be prepared inexpensively within the laboratory. Two such methods are – Polyethylene Glycol (Carbowax) Fixative 95% Ethyl alcohol = 50 ml Ether * = 50 ml Polyethylene glycol = 5 g Freshly made smears are placed on a flat surface and the slides are covered immediately by five or six drops of the fixative. Allow the slide to dry for 5 to 7 minutes or until an opaque, waxy film forms over the surface. 2. Diaphane Fixative 95% ethyl alcohol (3 parts) + Diaphane(2 parts)

50. Unless removed prior to staining, all coating fixatives will contaminate the staining solutions, particularly the hematoxylin. The water-soluble coating fixatives should be removed prior to staining by maintaining two separate dishes of 95% ethyl alcohol and leaving the slides in each dish for 5 to 10 minutes. The 95% ethyl alcohol used for washing off the coating fixative should be filtered or changed at least once each day, the number of times depending on the number of slides that are washed.

51. Special purpose fixatives Neutral buffered formalin Bouin’s solution - 1.2% (saturated) aqueous picric acid = 750 ml 37% to 40% Formaldehyde solution = 250 ml Glacial acetic acid = 50 ml Methanol acetic acid fixative - Equal volume of 20:1 methanol and acetic acid. Used when both cytologic evaluation and flow cytometry is desired on the same urine or bladder washing sample.

52. Balanced salt solutions / Normosol - Normosol is an excellent, low-cost alternative for short-term storage of FNA samples. 5. Formol alcohol Saccomano’s fixative – 50% alcohol + 2% Carbowax 1540 Carbowax infiltrates and occupies submicroscopic spaces, preventing cell collapse, and thus protects the cells during air drying first used by Saccomanno for prefixation of sputum but can be used for fluid specimens from other sites.

53. Carnoy’s fixative – 95% Ethanol = 60 ml Chloroform = 30 ml Glacial acetic acid = 10 ml This fixative will hemolyze red blood cells and, therefore, is useful for bloody specimens. However, shrinkage of the epithelial cells is greater than that observed in specimens fixed in 95% ethanol. Nuclear chromatin will be lost if the cell sample remains in Carnoy's fixative for longer than 15 minutes. This fixative must be prepared fresh when needed and discarded after each use. Carnoy's fixative loses its effectiveness on standing, and the chloroform can react with acetic acid to form hydrochloric acid.

54. References Bancroft's Theory and Practice of Histological Techniques, 7th Edition. Koss' Diagnostic Cytology and Its Histopathologic Bases, 5th Edition. Chatterjee, Shailja. “Artefacts in Histopathology.”  Journal of Oral and Maxillofacial Pathology : JOMFP  18.Suppl 1 (2014): S111–S116.  PMC .

55. Thank you