Fixation of tissues - Clinical Tree

Acknowledgment

We would like to thank all the previous contributors to this chapter for their scientific input and Dr Catherine Cannet for her review of the chapter for this edition and her updates.

Introduction

This chapter discusses the basics of fixation, alongside the advantages and disadvantages of specific fixatives. It also provides some of the formulas for these fixatives currently used in pathology, histology 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 mechanisms 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 several 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 features ( ). The development of specific fixatives has usually been empirical, although much of the understanding of the mechanisms of fixation has been based upon information obtained from leather tanning and vaccine production. In order to visualize the microanatomy of stained tissue sections, the original microscopic relationships between cells, cellular components (e.g. the cytoplasm and nuclei) and the extracellular material must be maintained with little disruption to the organization of the tissue. The local chemical composition of the tissue must also be maintained. Many tissue components are soluble in aqueous acid or other liquid environments and to reliably view the microanatomy and microenvironment of these tissues the soluble components must not be lost during fixation and tissue processing. Minimizing the loss of cellular components 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, lysosomes 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 damaged. 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 ( ). Various methods of fixation always produce some artifacts in the appearance of tissue on staining. However, for diagnostic pathology it is important that such artifacts 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 microorganisms. This results in one view of a dynamically changing, viable tissue ( ). The fixative should also prevent the subsequent breakdown of the tissue or molecular features by enzymatic 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 chemically altered tissues. Fixation interacts with all phases of processing and staining from dehydration to staining of tissue sections using histochemical, enzymatic or immunohistochemical stains ( ). It follows that any stained tissue section, produced after specific fixation combined with tissue processing, is a compromise 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 ( ). In diagnostic pathology, the fixative of choice for most pathologists has been 10% neutral buffered formalin ( ).

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 ( ). However, from a clinical perspective the advent of heat-induced epitope retrieval methods, instigated in the early 1990s, have overcome many of these limitations ( ). Similarly, the analysis of mRNA and DNA from formalin-fixed, paraffin-embedded tissue has been problematic ( ). 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 paraffin 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 soluble 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 ( ). 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 recovery of macromolecules including proteins, mRNA, and DNA from fixed and paraffin-embedded tissues without extensive biochemical modifications.

Other important characteristics of an ideal fixative 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 compatible with modern automated tissue processors. It should be readily disposable or recyclable, support long term tissue storage to give excellent microtomy of paraffin blocks and should be cost effective ( ).

Types of fixation

Fixation of tissues can be accomplished by physical and/or chemical methods. Physical methods, e.g. heating, microwaving and freeze-drying are independent processes and not used commonly in the routine practice of medical or veterinary pathology, anatomy and histology. The exception is the use of dry heat fixation of microorganisms prior to Gram staining. Most methods of fixation used in the processing of tissue for histopathological diagnoses rely on chemical fixation carried out by liquid fixatives. Reproducibility of the microscopic appearances of tissues after H&E staining is the prime requirement of the fixatives used for diagnostic pathology. Methods of fixation used in research protocols include the use of vapors and rarely, when fixation of a whole animal is needed, the perfusion of the animal’s vascular system with a fixative ( ).

Several chemicals, or their combinations, can act as good fixatives and accomplish many of the stated goals of fixation. Some fixatives add covalent reactive groups which may induce cross-links between proteins, individual protein moieties within nucleic acids and between nucleic acids and proteins ( ). The best examples of such ‘cross-linking fixatives’ are formaldehyde and glutaraldehyde.

Another approach to fixation is to use agents which remove free water from tissues and precipitate and coagulate the proteins. Examples of these dehydrants include ethanol, methanol and acetone. These agents denature proteins by breaking the hydrophobic bonds responsible for maintaining the tertiary structure of proteins. Other fixatives, e.g. acetic acid, trichloroacetic acid, mercuric chloride and zinc acetate act by denaturing proteins and nucleic acids through changes in pH or via salt formation.

Some fixatives are mixtures of reagents and are referred to as compound fixatives, e.g. alcoholic formalin fixes tissues in two ways: firstly, by adding covalent hydroxymethyl groups and cross-linking proteins and secondly, by coagulation and dehydration.

Physical methods of fixation

Heat fixation

This is the simplest form of fixation. Boiling or poaching an egg precipitates the proteins and, on cutting, the yolk and egg white can be identified separately. Each component is less soluble in water after heat fixation than the same component of a fresh egg. Picking up a frozen section on a warm microscope slide, both attaches the section to the slide and partially fixes it by heat and dehydration. Even though adequate morphology could be obtained by boiling tissue in normal saline, heat is primarily used to accelerate other forms of fixation as well as the other steps of tissue processing.

Microwave fixation

Microwave heating can reduce times for fixation of some gross specimens and histological sections from more than 12 hours to less than 20 minutes ( ). Microwaving tissue in formalin results in the production of large amounts of dangerous, potentially explosive vapors. In the absence of a hood for extraction or a microwave processing system designed to handle these vapors, this may cause safety problems. Commercial glyoxal-based fixatives which do not form vapors when heated at 55°C have been introduced as an efficient method of microwave fixation.

Freeze-drying and freeze substitution

Freeze-drying is a useful technique for studying soluble materials and small molecules. Tissues are cut into thin blocks, immersed in liquid nitrogen and the water removed in a vacuum chamber at −40°C. The tissue can be post-fixed with formaldehyde vapor. In freeze substitution, specimens are immersed in fixatives, e.g. acetone or alcohol at −40°C, this slowly removes water through dissolution of ice crystals and the proteins are not denatured. Bringing the temperature gradually up to 4°C will complete the fixation process ( ). These methods of fixation are used primarily in the research environment and are rarely used in the clinical laboratory setting.

Chemical fixation

This utilizes organic or non-organic solutions to maintain adequate morphological preservation. Chemical fixatives can be considered as members of three major categories: coagulant, cross-linking, and compound ( ).

Coagulant fixatives

Both organic and non-organic solutions may coagulate proteins making them insoluble. Cellular architecture in vivo is maintained primarily by lipoproteins and fibrous proteins such as collagen. Coagulating these proteins maintains tissue histomorphology at the light microscope level. Unfortunately, because coagulant fixatives result in cytoplasmic flocculation and poor preservation of mitochondria and secretory granules, these fixatives are not useful in ultrastructural analysis.

Dehydrant coagulant fixatives

The most commonly used in this group are alcohols (e.g. ethanol, methanol) and acetone. Methanol is closer to the structure of water than ethanol. Ethanol therefore competes more strongly than methanol in the interaction with hydrophobic areas of molecules and coagulant fixation begins at a concentration of 50–60% for ethanol but 80% or more for methanol ( ). The removal and replacement of free water from tissue by any of these agents has several potential effects on proteins within the tissue. Water molecules surround hydrophobic areas of proteins and, by repulsion, force hydrophobic chemical groups into closer contact with each other stabilizing hydrophobic bonding. By removing water, the opposite principle weakens hydrophobic bonding. Similarly, molecules of water participate in hydrogen bonding in hydrophilic areas of proteins, and therefore removal of water destabilizes this hydrogen bonding. Together, these changes act to disrupt the tertiary structure of proteins. Additionally, with the water removed the structure of the protein may become partially reversed, with hydrophobic groups moving to the outside surface of the protein. Once the tertiary structure of a soluble protein has been modified, the rate of reversal to a more ordered soluble state is slow and most proteins after coagulation remain insoluble even if returned to an aqueous environment.

Disruption of the tertiary structure of proteins (i.e. denaturation) changes their physical properties, potentially causing insolubility and the loss of function. Even though most proteins become less soluble in organic environments, up to 13% of protein may be lost, e.g. with acetone fixation ( ). Factors which influence the solubility of macromolecules include:

Temperature, pressure, and pH.
Ionic strength of the solute.
The salting-in constant, which expresses the contribution of the electrostatic interactions.
The salting-in and salting-out interactions.
The types of denaturing reagents ( ).

Alcohol denatures protein differently depending on the choice and concentration of alcohol, the presence of organic and non-organic substances and the pH and temperature of fixation.

The protein denaturing effect of ethanol is > phenols > water and polyhydric alcohols > monocarboxylic acids > dicarboxylic acids ( ).

Other types of coagulant fixative

The charges on the ionizable side chains, e.g. –NH ₂ → NH ₃ ⁺ and COO ⁻ → COOH, are changed using acid coagulants such as picric and trichloroacetic acid by the disruption of electrostatic and hydrogen bonding. These acids may also insert a lipophilic anion into a hydrophilic region and 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. Trichloroacetic acid (Cl ₃ CCOOH) can penetrate hydrophobic domains of proteins and the anion produced (–C–COO ⁻ ) reacts with charged amine groups. This interaction precipitates proteins and extracts nucleic acids. Picric acid or trinitrophenol dissolves slightly in water to form an acid solution (pH 2.0). In reactions it forms salts with basic groups of proteins causing the proteins to coagulate. If the solution is neutralized, the precipitated protein may re-dissolve. Picric acid fixation produces brighter staining, but the low pH solution may cause hydrolysis and the loss of nucleic acids.

Non-coagulant cross-linking fixatives

Several chemicals were selected as fixatives secondary to their potential actions of forming cross-links both within and between proteins and nucleic acids. Cross-linking may not be a major mechanism with short times of fixation and therefore ‘covalent additive fixatives’ may be a better name for this group. Examples include formaldehyde, glutaraldehyde and other aldehydes, e.g. chloral hydrate and glyoxal, as well as metal salts, e.g. mercuric and zinc chloride, and other metallic compounds, e.g. osmium tetroxide. Aldehyde groups are chemically and biologically reactive and are responsible for many histochemical reactions, e.g. the argentaffin reaction ( ).

Formaldehyde fixation

Formaldehyde, as 10% neutral buffered formalin (NBF) is the most common fixative used in diagnostic pathology. Pure formaldehyde is a vapor which, when completely dissolved in water forms a solution containing 37–40% formaldehyde and this aqueous solution is ‘formalin’. The usual ‘10% formalin’ used in the fixation of tissues is a 10% solution of formalin, which contains approximately 4% weight to volume of formaldehyde.

The reactions of formaldehyde with macromolecules are numerous and complex. and ) meticulously identified most of the reactions of formaldehyde with amino acids and proteins using simple chemistry. In an aqueous solution, formaldehyde forms methylene hydrate, a methylene glycol as the first step in fixation ( ).

H_{2} C = O + H_{2} O \to {HOCH}_{2} OH

$H_{2} C = O + H_{2} O \to {HOCH}_{2} OH$

Methylene hydrate reacts with several side chains of proteins to form reactive hydroxymethyl side groups (–CH ₂ –OH). When the current relatively short fixation times are used with 10% neutral buffered formalin (hours to days), the formation of hydroxymethyl side chains is the primary and characteristic reaction and the formation of actual cross-links may be rare.

Formaldehyde also reacts with nuclear proteins and nucleic acids ( ). It penetrates between nucleic acids and proteins and stabilizes the nucleic acid-protein shell, also modifying nucleotides by reacting with the free amino groups as it does with proteins. In naked and free DNA, the cross-linking reactions are believed to start at adenine-thymidine rich regions and cross-linking increases with rising temperatures ( ). Formaldehyde reacts with C=C and –SH bonds in unsaturated lipids but does not interact with carbohydrates ( ).

The side chains of peptides or proteins which are most reactive with methylene hydrate have the highest affinity for formaldehyde; these include lysine, cysteine, histidine, arginine, tyrosine and the reactive hydroxyl groups of serine and threonine ( Table 4.1 ) ( ).

Table 4.1

Action of major single or combination fixatives

Category of fixative	Dehydrants	Aldehyde cross-linkers	Combination mercuric chloride with formaldehyde or acetic acid	Osmium tetroxide	Picric acid plus formalin and acetic acid	Combination alcohols plus formalin
Examples of category	Ethanol Methanol Acetone	Formaldehyde Glutaraldehyde	Zenker’s B5	Post-fixation after glutaraldehyde	Bouin’s	Alcoholic formalin
Effect on proteins	Precipitates without chemical addition	Cross-linkers: adds active hydroxymethyl groups to amines, amides, some reactive alcohols, and sulfydryl groups; cross-links amine/amide or sulfydryl side chains of proteins	Additive plus coagulation	Additive cross-links; some extraction, some destruction	Additive and non-additive coagulant, some extraction	Additive plus precipitation
mRNA/DNA	Slight	Slowly cross-links; slightly extracts	Coagulation	Slight extraction	No action	Slight
Lipids	Extensive extraction	No action	No action	Made insoluble by cross-links with double bonds	No action	Extensive extraction
Carbohydrates	No action	None on pure carbohydrates; cross-linking of glycoproteins	No action	Slight oxidation	No action	No action
Quality of H&E staining	Satisfactory	Good	Good	Poor	Good	Good
Effect on ultrastructure (organelles)	Destroys ultrastructure, including mitochondria, proteins, coagulates	Preservation with NBF good, excellent with glutaraldehyde and adequate to good with Carson-Millonig’s	Poor preservation	Used for visualization of membranes	Poor – tends to destroy membranes	Poor
Usual formulation	70–100% solution or in combination with other types of fixative	Formaldehyde (37%) – 10% V/V aqueous solution buffered with phosphates to pH 7.2–7.4. Glutaraldehyde – 2% buffered to pH 7.4	Mercuric chloride combined either with acetic acid plus dichromate or with formaldehyde plus acetate	1% solution buffered to pH 7.4	Aqueous picric acid, formalin, glacial acetic acid	10% formaldehyde (37%) with 90% ethanol
Important variables/issues	Time, specimen thickness – should be used only for small or thin specimens	Time, temperature, pH, concentration/ specimen thickness	Toxic	Extremely toxic	Mitochondria and integrity of nuclear membrane destroyed; not appropriate for some stains	Time, specimen dimensions. Note good fixative for renal tissues
Special uses	Preserves small non-lipid molecules such as glycogen; preserves enzymatic activity	General all-round fixative; best for ultrastructure if used with osmium tetroxide post-fixation	Excellent for hematopoietic tissues	Ultrastructural visualization of membranes; lipids on frozen sections	Mordant for connective tissue stains (trichrome)	Good general fixative; good for specific immunohistochemical reactions and good to detect lymph nodes in fatty tissue; removes fats from tissue

reported that one of the most important cross-links in ‘over-fixation’, i.e. in tanning, is between lysine and the amide group of the protein backbone but again with the current shorter fixation times this is unlikely to occur ( ).

Reversibility of formaldehyde-macromolecular reactions

The reactive groups may combine with hydrogen groups or with each other to form methylene bridges. If the formalin is washed away, reactive groups may rapidly return to their original states, but any bridging which has already occurred may remain. Washing for 24 hours removes approximately half of the reactive groups and after 4 weeks up to 90% are removed ( ). This suggests that actual cross-linking is a relatively slow process and, most ‘fixation’ with formaldehyde prior to tissue processing in diagnostic pathology stops with the formation of reactive hydroxymethyl groups.

During long term storage in formalin, the reactive groups may be oxidized to the more stable groups (e.g. acids –NH–COOH) which are not easily removed by washing in water or alcohol. Returning the specimen to water or alcohol following fixation therefore reduces the further fixation of the specimen because the reactive groups produced by the initial reaction with formalin may reverse and be removed. Although it was initially thought that cross-linking was the most important process in the fixation of tissue for biological uses (based on the limited number of cross-links over short periods of fixation), it is likely that the formation of these hydroxymethyl groups actually denatures macromolecules and renders them insoluble. As these washing experiments have not been reproduced, the actual mechanisms and their importance in fixation by formaldehyde remain uncertain.

Over-fixation of tissue may also be partially corrected by soaking the tissue in concentrated ammonia plus 20% chloral hydrate ( ). Fraenkel-Conrat and his colleagues noted that the addition and condensation reactions of formaldehyde with amino acids and proteins were unstable and could be reversed easily by dilution or dialysis ( ).

The principal type of cross-link in short term fixation is thought to be between the hydroxymethyl group on a lysine side chain and arginine (through secondary amino groups), asparagine and glutamine (through secondary amide groups) or tyrosine (through hydroxyl groups) ( ). For example, a lysine hydroxymethyl amine group can react with an arginine group to form a lysine–CH ₂ –arginine cross-link. Similarly, a tyrosine hydroxymethyl amine group can bind with a cysteine group to form a tyrosine–CH ₂ –cysteine cross-link. Each of these cross-links between macromolecules has a different degree of stability which can be modified by the temperature, pH and the type of environment surrounding and permeating the tissue ( ). The time to saturation of human and animal tissues with active groups by formalin is approximately 24 hours, but cross-linking may continue for many weeks ( ).

When formaldehyde dissolves in an unbuffered aqueous solution, it forms an acid solution (pH 5.0–5.5) because 5–10% of commercially available formaldehyde is formic acid. Acid formalin may react more slowly with proteins than NBF because the amine groups become charged, e.g. –N ⁺ H ₃ . In solution, this requires a much lower pH than 5.5. However, the requirement for a lower pH to produce –N ⁺ H ₃ groups may not be equivalent to that required in peptides. Acid formalin also preserves immunorecognition better than NBF ( ). Indeed, the success of early immunocytochemistry methods to demonstrate immunoglobulins in paraffin-processed tissue sections probably relied on the fixation of the tissues in acid formalin ( ). The disadvantage of using acid formalin for fixation is the formation of a brown/black pigment with degraded hemoglobin. This heme-related pigment which forms in tissue, is not a problem unless patients have a blood abnormality, e.g. sickle cell disease or malaria.

Formaldehyde primarily preserves peptide-protein bonds and the general structure of cellular organelles. It can interact with nucleic acids but has little effect on carbohydrates and preserves lipids if the solutions contain calcium ( ).

Glutaraldehyde fixation

Less is known about the biological reactions and effects of glutaraldehyde compared to formaldehyde as it has not been used as widely in biological applications. Glutaraldehyde is a bifunctional aldehyde which probably combines with the same reactive groups as formaldehyde. In aqueous solutions glutaraldehyde polymerizes forming cyclic and oligomeric compounds ( ); it is also oxidized to glutaric acid. It requires storage at 4°C and a pH of approximately 5 for stability ( ).

Unlike formaldehyde, glutaraldehyde has an aldehyde group at both ends of the molecule. Following each reaction of the first group, an unreacted aldehyde group may be introduced into the protein and these groups can act to further cross-link the protein. Alternatively, the aldehyde groups may react with a wide range of other histochemical targets which include antibodies, enzymes or proteins. The reaction of glutaraldehyde with an isolated protein such as bovine serum albumin, is fastest at pH 6–7 and results in more cross-linking than formaldehyde ( ). Cross-linking is irreversible and withstands acids, urea, semicarbazide and heat ( ). Similar to formaldehyde, reactions with lysine are the most important for forming cross-links.

Extensive cross-linking by glutaraldehyde results in better preservation of the ultrastructure, but this method of fixation negatively affects immunohistochemical methods and slows the penetration by the fixative. Any tissue fixed in glutaraldehyde must be small (0.5 mm maximum) and, unless the aldehyde groups are blocked, increased background staining will result ( ). Glutaraldehyde does not react with carbohydrates or lipids unless they contain free amino groups which are found in some phospholipids ( ). At room temperature glutaraldehyde does not cross-link nucleic acids in the absence of nucleohistones, but it may react with nucleic acids at or above 45°C ( ).

Osmium tetroxide fixation

Osmium tetroxide (OsO ₄ ) is a toxic solid which is soluble in water as well as non-polar solvents. It can react with hydrophilic and hydrophobic sites including the side chains of proteins where it potentially can cause cross-linking ( ). The reactive sites include sulfydryl, disulfide, phenolic, hydroxyl, carboxyl, amide and heterocyclic groups. Osmium tetroxide is known to interact with nucleic acids, specifically the 2,3-glycol moiety in terminal ribose groups and the 5,6 double bonds of thymine residues. Nuclei fixed in OsO ₄ and dehydrated with alcohol may show prominent clumping of DNA. This unacceptable artifact can be prevented by pre-fixation with potassium permanganate (KMnO ₄ ), post-fixation with uranyl acetate or by adding calcium ions and tryptophan during fixation ( ). The reaction of OsO ₄ with carbohydrates is variable ( ). Large proportions of proteins and carbohydrates are lost from tissues during osmium fixation. This may be due to the superficial penetration of OsO ₄ (<1 mm) into tissues, or its slow rate of reaction. In electron microscopy this loss is minimized by initial fixation of tissue in glutaraldehyde.

The best characterized reaction of osmium is its reaction with unsaturated bonds within lipids and phospholipids. Osmium, in this reaction, alters from the +8 valence state to the +6 valence state, which is colorless. If two unsaturated bonds are close together there may be cross-linking by OsO ₄ . Although the complex is colorless at this point, the typical black staining of membranes expected from fixation with osmium requires the production of osmium dioxide (OsO ₂ ·2H ₂ O). Osmium dioxide is black, electron dense and insoluble in aqueous solution; it precipitates as the above unstable compounds break down and becomes deposited on cellular membranes. The breakdown of osmium +6 valence complexes to osmium dioxide (+4 valence state) is facilitated by a reaction with solutions of ethanol.

In addition to its use as a secondary fixative for electron microscope examinations, OsO ₄ can be used to stain lipids in frozen sections. Osmium tetroxide fixation causes tissue swelling which is reversed during the dehydration steps. Swelling can also be minimized by adding calcium or sodium chloride to osmium-containing fixatives ( ).

Cross-linking fixatives for electron microscopy

Cell organelles, e.g. cytoplasmic and nuclear membranes, mitochondria, membrane-bound secretory granules, smooth and rough endoplasmic reticula, need to be preserved carefully for electron microscopy. The lipids in these structures are extracted by many dehydrating fixatives, e.g. alcohols, and for ultrastructural examination it is therefore important to use a fixative which does not remove lipids. Strong cross-linking fixatives are preferred, e.g. glutaraldehyde, a combination of glutaraldehyde and formaldehyde or Carson’s modified Millonig’s, followed by post-fixation in an agent such as OsO ₄ which further stabilizes and emphasizes membranes.

Mercuric chloride fixatives

Historically, mercuric chloride was favored for its ability to enhance the staining properties of tissues, particularly with the trichrome stains. However, it is now rarely used in the clinical laboratory due to the health and safety issues of mercury-containing fixatives and, the reduced reliance on ‘special stains’. A further major disadvantage of mercuric chloride fixation is the formation of intense black precipitates of mercuric pigment in the tissue which gives them inferior value for immunohistochemical and molecular studies. In recently fixed tissues, these precipitates can be removed by a Lugol’s iodine step in the staining procedure, followed by bleaching of the section in sodium hypochlorite solution (Hypo). However, this is not effective on mercuric chloride fixed tissues which have been stored for a number of years as paraffin blocks. In these tissues, retrospective analysis by immunohistochemistry and molecular techniques is unreliable due to the formation of much larger aggregates of mercuric pigment which cannot be removed by Lugol’s iodine. The chemistry of fixation using mercuric chloride is not well understood but it is known that mercuric chloride reacts with ammonium salts, amines, amides, amino acids and sulfydryl groups to harden tissues. It is especially reactive with cysteine, forming a dimercaptide ( ) which acidifies the solution:

\begin{matrix} Sulfydryl - 2 (R - S - H) + {HgCl}_{2} ⇌ \\ {(R - S)}_{2} - Hg + 2 H^{+} + 2 {Cl}^{-} \end{matrix}

$\begin{matrix} Sulfydryl - 2 (R - S - H) + {HgCl}_{2} ⇌ \\ {(R - S)}_{2} - Hg + 2 H^{+} + 2 {Cl}^{-} \end{matrix}$

If only one cysteine is present, a reactive group of R–S–Hg–Cl is likely.

Mercury-based fixatives are toxic and all 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-containing chemicals also pose an environmental disposal problem.

These fixatives penetrate slowly, so specimens must be thin, and mercury and acid formaldehyde hematein pigments may deposit in tissue after fixation. Mercury fixatives ( ) are no longer used routinely, except, e.g. B5, by some laboratories for fixing hematopoietic tissues. A potential replacement for mercuric chloride is zinc sulfate. Special formulations of zinc sulfate in formaldehyde replacing mercuric chloride in B5 may give better nuclear detail than formaldehyde alone and improve tissue penetration ( ).

Special fixatives

Dichromate and chromic acid fixation

Chromium trioxide dissolves in water to produce an acidic solution of chromic acid with a pH of 0.85 and this is a powerful oxidizing agent which produces aldehyde from the 1, 2-diglycol residues of polysaccharides. These aldehydes can react with histochemical stains, e.g. PAS and argentaffin/argyrophil and should increase the background of immunohistochemical staining ( ).

Actual chromic salts, i.e. chromium ions in +3 valence state, may destroy animal tissues ( ), but chromium ions in their +6 state coagulate proteins and nucleic acids. The fixation and hardening reactions are not fully understood, but probably involve the oxidation of proteins which varies in strength depending upon the pH of the fixative, and the interaction of the reduced chromate ions directly in cross-linking proteins ( ). Chromium ions specifically interact with the carboxyl and hydroxyl side chains of proteins and chromic acid interacts with disulfide bridges and attacks lipophilic residues such as tyrosine and methionine ( ). Fixatives containing chromate at a pH of 3.5–5.0 make proteins insoluble without coagulation. Chromate is reported to make unsaturated but not saturated lipids insoluble upon prolonged (>48 hours) fixation and hence mitochondria are well preserved by these fixatives.

Dichromate-containing fixatives have primarily been used to prepare neuroendocrine tissues for staining, especially normal adrenal medulla and its related tumors, e.g. pheochromocytomas. However, reliance on the chromaffin reaction used to identify chromaffin granules following dichromate fixation is now being replaced by immunohistochemistry with a range of neuroendocrine markers including chromagranin A and synaptophysin ( ).

Fixatives for DNA, RNA and protein analysis

conducted a comprehensive analysis of 25 fixative compounds, many reputed to provide improved preservation of DNA, RNA and proteins in tissues for immunocytochemical analysis, whilst at the same time ensuring optimal morphological preservation. These compounds included the commercially available HEPES-glutamic acid buffer mediated Organic Solvent Protection Effect (HOPE) fixative, the reversible cross-linker dithiobis [succinimidyl propionate] (DSP) for immunocytochemistry and expression profiling, and zinc-based fixatives. They concluded that a novel zinc formation, Z7, containing zinc trifluoroacetate, zinc chloride and calcium acetate was significantly better than the standard zinc-based fixative, Z2 and NBF for DNA, RNA and antigen preservation. DNA and RNA fragments up to 2.4kb and 361bp in length respectively, were detected by a polymerase chain reaction (PCR), reverse transcriptase PCR and real-time PCR in the Z7 fixed tissues, also allowing protein analysis using 2D electrophoresis. Nucleic acids and protein were found to be stable over a period of 6–14 months. The fixative is also less toxic than formaldehyde formulations. Whilst this Z7 fixative has shown great promise, it should be borne in mind that fixation in NBF will also allow the extraction of similarly sized fragments of DNA and RNA for analysis by PCR-based technologies within the same time frame.

Metallic ions as a fixative supplement

Several metallic ions have been used as aids in fixation, including Hg ²⁺ , Pb ²⁺ , Co ²⁺ , Cu ²⁺ , Cd ²⁺ , [UO ₂ ] ²⁺ , [PtCl ₆ ] ²⁺ and Zn ²⁺ . 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. This does however depend upon the pH of the formaldehyde, as well as the zinc formaldehyde ( ).

Compound fixatives

Pathologists use formaldehyde-based fixatives to ensure reproducible histomorphometric patterns. Other agents may be added to formaldehyde to produce specific effects which are not possible with formaldehyde alone. The dehydrant ethanol, for example, can be added to formaldehyde to produce alcoholic formalin. This combination preserves molecules such as glycogen and results in less shrinkage and hardening than pure dehydrants.

Compound fixatives are useful for specific tissues, e.g. alcoholic formalin for fixation of some fatty tissues, such as breast, in which the preservation of the lipid is not important. Additionally, the fixation of gross specimens in alcoholic formalin may aid the identification of lymph nodes embedded in fat (see also page 55 ). Some combined fixatives, including alcoholic formalin, are good at preserving antigen immunorecognition, but non-specific staining or background staining in immunohistochemical procedures can be increased. Unreacted aldehyde groups in glutaraldehyde-formaldehyde fixation, for example, may increase background staining and alcoholic formalin may cause non-specific staining of myelinated nerves ( ).

Factors affecting the quality of fixation

Buffers and pH

The effect of pH on fixation with formaldehyde may be profound depending upon the applications to which the tissues will be exposed. In a strongly acidic environment, the primary amine target groups (–NH ₂ ) attract hydrogen ions and become unreactive (–NH ⁺ ₃ ) to the hydrated formaldehyde (methylene hydrate or methylene glycol), and carboxyl groups (–COO ⁻ ) lose their charges (–COOH). This may affect the structure of proteins. Similarly, the hydroxyl groups of alcohols (–OH) including serine and threonine may become less reactive in a strongly acidic environment. The extent of formation of reactive hydroxymethyl groups and cross-linking is reduced in unbuffered 4% formaldehyde ( ) which is slightly acidic ( ), because the major methylene cross-links are between lysine and the free amino group on the side chains.

The decrease in the effectiveness of formaldehyde fixation and hence cross-linking in the slightly acid environment has led some authors to suggest that unbuffered formalin is a better fixative than NBF for the immunorecognition of many antigens ( ). This aided the detection of antigens prior to the advent of heat-induced epitope retrieval methods in immunocytochemistry. However, minimal delay in effectively fixing labile antigens, such as the estrogen receptor, is vital in the immunohistochemical testing for a range of clinically important prognostic and predictive biomarkers. Whilst formaldehyde fixation remains the recommended method for optimal preservation of morphological features, proteins and nucleic acids in a clinical environment, the most reliable way of achieving optimal formalin fixation is through its buffering at pH 7.2–7.4, i.e. NBF.

At the acidic pH of unbuffered formaldehyde, hemoglobin metabolic products are chemically modified to form a brown-black, insoluble, crystalline, birefringent pigment. This pigment forms at a pH less than 5.7 and the extent of its formation increases in the pH range 3.0 to 5.0. Formalin pigment is easily recognized and should not affect diagnoses except in patients with large amounts of hemoglobin breakdown products secondary to hematopoietic diseases. The pigment is removed easily with an alcoholic solution of picric acid. Using NBF avoids the formation of formalin pigment and it is used as the preferred formaldehyde- based fixative.

Acetic and other acids work mainly through lowering pH and disrupting the tertiary structure of proteins. Buffers are used to maintain the optimum pH. The choice of a specific buffer depends on the type of fixative and analyte. Commonly used buffers are phosphate, cacodylate, bicarbonate, Tris and acetate. It is necessary to use low salt, buffered formalin in the new complex tissue processors in order to keep the machine ‘clean’ and reduce problems in its operation.

Duration of fixation and the size of specimens

The factors which govern diffusion of a fixative into tissue were investigated by . He found that the depth in mm, d, reached by a fixative is directly proportional to the square root of the duration of fixation in hours, t, and expressed this relation as:

d = k \sqrt{t}

$d = k \sqrt{t}$

The constant, k, is the coefficient of diffusability which is specific to each fixative. Examples are 0.79 for 10% formaldehyde, 1.0 for 100% ethanol and 1.33 for 3% potassium dichromate ( ). Thus, the time of fixation is approximately equal to the square of the depth to which the fixative must penetrate and most fixatives, such as NBF will penetrate tissue to the depth of approximately 1 mm in one hour, e.g. for a 10 mm sphere, the fixative will not penetrate to the center until (5) ² , 25 hours of fixation. It is important to note that the components of a compound fixative will penetrate the tissue at different rates so it is better if these fixatives are used with thin specimens.

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 allowing penetration of the fixative in all directions. Additionally, unfixed gross specimens which are to be cut and stored in fixative prior to processing, should not be thicker than 5 mm. When surgical specimens are to be processed to paraffin blocks the time of penetration by the fixative is more critical. Specific issues related to the processing of tissues have been reviewed by and .

Fixation proceeds slowly – the period between the formation of reactive hydroxymethyl groups and the formation of a significant number of cross-links is unknown. Ninety percent of reactive groups can be removed by 4 weeks of washing ( ), confirming that cross-linking is not a rapid process and may require weeks for the completion of potential bonds.

Proteins inactivate fixatives, especially in blood or bloody fluids, so these gross specimens should 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. Currently in some laboratories, thin specimens may be fixed in NBF for only 5–6 hours, including the short time of fixation in tissue processors. The extent of the formation of cross-links during such rapid NBF fixation is uncertain. Consequently, the formation of hydroxymethyl groups may predominate as opposed to the more resilient cross-linking. 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 fixative, e.g. formaldehyde. However, studies investigating the time taken to adequately fix clinical cases of breast cancer tissue for subsequent immunohistochemical detection of estrogen receptors illustrate that this practice can be detrimental to the optimal preservation of important antigens, and should be avoided. found that 6–8 hours was the minimum time required to adequately fix breast tissue for immunohistochemical testing of estrogen receptors, regardless of the size and type of the specimen. Consequently, the guidelines for estrogen receptor and progesterone receptor testing produced by the American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) recommend this minimal fixation time in neutral buffered formalin for all clinical breast cancer specimens ( )

You're Reading a Preview

Become a Clinical Tree membership for Full access and enjoy Unlimited articles

Become membership

If you are a member. Log in here