INTRODUCTION
Histological techniques are a series of processes by which the tissues are prepared for microscopic examination. It starts with fixation and continues with dehydration, clearing, embedding, cutting and staining.
When the tissues are removed from the body (e.g. surgical excision) or blood supply is cut off; tissues begin to decompose. This is due to lack of oxygen and other essential metabolites and also from the accumulation of carbon dioxide and other toxic metabolites in the cells and due to activation of different autolytic enzymes. Some tissues are decomposed rapidly whereas others are slow to decompose. The rapidity is proportional to the natural metabolic activity of the tissue. This is the basis of rapid decomposition in liver, pancreas, convoluted tubules in kidney.
To minimize the decomposition and to preserve as nearly as possible the natural activity of the cells; the tissues are put in a suitable fixative (usually 10–20 times of volume of surgical specimens). Fixation is defined as a complex series of chemical and physical events which prevents or at least minimizes tissue decomposition and it differs for the different groups of chemical substances found in the tissues. There are other reasons of fixation. In most of the cells, there is an outer complex membrane containing the fluid protoplasm. The protoplasm is a mixed, true and colloidal solution of carbohydrates, proteins, lipids, salts, organic acids and enzymes. Many of these substances would have been lost if tissues had not been fixed.
The aims of fixation are:
- Prevention of tissue autolysis and bacterial attack
- To maintain the shape and volume of the tissues during subsequent procedures (e.g. clearing, embedding, etc.)
- To keep tissues as close to their natural state and to minimize change of natural color and appearance
- To prevent loss of tissue substances or rearrangement of tissue ingredients.
To achieve the aims of fixation, an ideal fixative should:
- Prevent autolysis and bacterial decomposition
- To maintain the shape and volume, natural color and appearance of the tissues
- Make the cellular components ‘fixed' after chemical and physical events of fixation and make cellular components insoluble to liquids during subsequent stages of processing
- It should be nontoxic, nonallergic and avoid excessive hardening of the tissues
- It allows enhanced staining of the tissues
- It should fix all the tissue components (carbohydrates, proteins, lipids) and prevents loss of any cellular components during next stages.
But, truly speaking, ideal fixative has not been found and search for it is still on. We commonly use fixatives which usually preserve protein (sacrificing carbohydrates, lipids) and conserve structures for routine purposes.
CLASSIFICATION OF FIXATIVES
- Aldehydes: Formaldehyde, glutraldehyde, acrolein.
- Protein denaturating agents: Acetic acid, methyl alcohol, ethyl alcohol.
- Physical agents: Microwave, heat.
- Other cross-linking agents: Carbodiimides.
- Miscellaneous: Picric acid, mercuric chloride.
REACTIONS OF FIXATIVES WITH PROTEINS
The most important reaction in tissue fixation is probably the reaction which stabilizes the proteins. Most commonly used fixatives have the property to form cross-link between proteins leading to gel formation. Soluble proteins get fixed with structural proteins (insoluble) and make the soluble protein insoluble. This process also gives some mechanical strength which allows subsequent procedure to take place.
Most fixatives used in laboratory are liquids/aqueous solutions, although vapor may be rarely used.
GENERAL PRINCIPLES OF FIXATION
- Amount of fixing fluid: Approximately 10–20 times the volume of the specimen, except for osmium tetroxide.
- Surgical specimens: Should be placed in fixative as soon as possible after removal from body. Bacteriological study is possible if done immediately.
- Autopsy specimen: Autopsy examination should be done after death without delay, if not possible then it should be kept in mortuary refrigerator at 4°C or arterial embalming should be done. Bacteriological or toxicological study not advisable after these procedures.
- Duration: Usually for 8 hours at room temperature when tissues are fixed with 10–20 times volume of 10% buffered formalin. But duration of 4 hours is sufficient if fixation is done with agitation. If temperature is raised to 45°C then fixation time may be shortened to 25–40% of usual time.
Aldehyde (Formaldehyde/10% Formalin/Glutaraldehyde)
The reactions between fixative and tissue proteins are usually mild with a short reaction time. Cross-links between proteins are formed and the reaction starts with basic amino acid lysine. Lysine amino acids which are on the exterior aspect of the protein molecule can only react.
In case of formaldehyde the reaction is usually reversible within the first 24 hours by an excess of water but glutaraldehyde causes rapid and irreversible reaction.
Aldehyde fixation may denature proteins to some extent apart from their primary role in forming cross-links. As most of the proteins are not denatured, tissues fixed with aldehyde may be used for immunohistochemistry (IHC), enzyme histochemistry and high resolution electron microscopy. But glutaraldehyde may cause a loss of up to 30% of alpha helix structure of protein in contrast to formaldehyde.
Cross-linkages between proteins and aldehydes can be measured by changes in molecular size, mechanical strength and viscosity. When two proteins are cross-linked, their molecular weight (MW) become doubled and it keeps on increasing as the polymerization (cross-linking of proteins) proceeds (Table 1).
ROUTINE FORMALIN FIXATIVES
This is a fixative which is most commonly used in histological laboratories. The commercially available solution contains 30–40% formaldehyde gas by weight and is called formalin. So, a 10% formalin fixative gives 4% formaldehyde gas for tissue fixation. Most laboratories prefer 10% buffered formalin or 10% formal saline as fixative (Table 2).
Chemical Composition of Different Formaldehyde Containing Fixatives
Formal Saline (10%)
- Water (preferably distilled): 900 mL.
- Sodium chloride: 9 g.
- Formalin (40% formaldehyde): 100 mL.
10% Formalin (4% Formaldehyde)
- Formalin (40% formaldehyde): 100 mL.
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Neutral Buffered Formalin (10%)
- Water (preferably distilled): 900 mL
- Formalin: 100 mL
- Sodium dihydrogen phosphate monohydrate: 4 g (NaH2PO4.H2O)
- Disodium hydrogen phosphate anhydrous: 6.5 g (Na2HPO4.).
REMOVAL OF FORMALIN PIGMENT
Kardasewitch's Method
- Put the histologic sections in water.
- Then place the sections in a mixture for 5 minutes to 3 hours (more the pigment, more the time required). The mixture contains:
- 70% ethyl alcohol: 100 mL.
- 28% ammonia water: 1–2 mL.
- After that take it out from the mixture and wash thoroughly in running tap water for 10–15 minutes.
Lillie's Method
- Keep the sections in water.
- Then place the sections in a mixture for 1–5 minutes. The mixture contains:
- Acetone: 50 mL.
- 3 volume hydrogen peroxide: 50 mL.
- 28% ammonia water: 1 mL.
- Wash in 70% alcohol (ethyl) for 1–2 minutes.
- Wash in running tap water for 10–15 minutes.
Picric Acid Method
- Put the sections in water.
- Place the sections in a saturated solution of picric acid for 5 minutes to 2 hours (depending on the amount of artifactual formalin pigments).
- Wash in running tap water for 10–15 minutes.
PROTEIN DENATURING AGENTS AS FIXATIVE
- Alcohol: This is used as 80–100% solution. It frequently shrinks and hardens the tissue. Does not preserve chromatin well but good for demonstrating glycogen, plasma cells, amyloid, iron and uric acid. Carnoy's fluid is generally used for specific purposes. Alcohol denatures and precipitates proteins probably by disrupting hydrogen bonds. Ethyl alcohol is used as a fixative for enzymes (Table 3).
- Acetic acid: It is used as 1–5% aqueous solution. It is good for nuclear fixation and has rapid penetration (so, fixation time is less). Disadvantage of this fixative is that it forms pigments if used with formalin. Also it causes hemolysis of RBCs.
Carnoy's Fixative
- Absolute alcohol: 60 mL.
- Chloroform: 30 mL.
- Glacial acetic acid: 10 mL.
Oxidizing Agent as Fixative
Chromium salts in water (aqueous solution) form Cr-O-Cr complexes. This leads to breakage of internal salt linkages of proteins and increases the activity of basic groups leading to enhanced acidophilia. Oxidizing agents react with proteins and osmium tetroxide to form cross-links with proteins.
- Osmic acid: It is used as 1–2% solution. It is very powerful oxidizing agent and is very expensive. So it 4cannot be used with formalin or alcohol. It fixes and stains fat. It is sometimes used for special cytologic methods, e.g. demonstration of Golgi bodies.
Table 3 Advantages and disadvantages of alcohol fixative AdvantagesDisadvantages- Methyl alcohol (80–100%) is excellent fixative for smears
- Ethyl alcohol is used as a fixative for enzymes
- Carnoy's fixative is used for urgent biopsy (paraffin processing within 5 hours)
- Good fixative to demonstrate glycogen, alkaline phosphatise, etc.
- Should be used at 0°C or cooler, otherwise causes marked shrinkage
- Distorts morphology and hardens the tissue
- Contraindicated for lipid study
- Although glycogen can be demonstrated it causes polarization (streaming of protoplasm to one pole of the cell) of glycogen granules
- Potassium dichromate: This is used as 2–4% aqueous solution. Compared to osmic acid, this is a weak oxidizing agent. It is a poor nuclear fixative as it dissolves nuclear chromatin and causes moderate shrinkage. But it is a good cytoplasmic fixative.
Orth's Fluid
- 2.5% potassium dichromate, K2Cr2O7 (aqueous): 100 mL.
- Sodium sulfate (optional): 1 g.
- Formalin (add just before using): 10 mL.
Regaud's (Moller's) Fluid
- 3% potassium dichromate: 80 mL.
- Formalin (add just before using): 20 mL.
Physical Agents as Fixatives
- Microwave: The optimum temperature for fixation is 45–55°C. Overheating (&65°C) may cause pyknotic nuclei, vacuolation and cytoplasmic over-staining whereas under-heating (<45°C) causes poor quality of tissue sections. It may be used for rapid fixation of tissues as required in urgent cardiac biopsies. Microwave fixed tissue may be used for electron microscopy aftert post-fixation in osmium tetroxide. Mode of action is through protein denaturation (no cross-linking of proteins as in formalin/aldehyde).
- Heat: Like the microwave controlled heat may be used as fixative.
Other Cross-linking Agents
They give alternative or improved fixation for electron microscopy or for gastrointestinal hormones demonstration. As for example, carbodiimides which were described as fixative by Hassel and Hand in 1974.
MERCURY FIXATIVES
Its tissue penetration is poor and cause tissue shrinkage. So, it is usually combined with other fixatives as a mixture. But tissue fixed with mercuric chloride usually results in black precipitates of mercury. This precipitates can be removed by keeping tissue sections in 0.5% iodine solution in 70% alcohol for 5–10 minutes. Then the sections are washed in water and decolorized in 5% sodium thiosulfate for 5 minutes. Again sections are washed in running water for 2–5 minutes (Table 4).
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Zenker's Fluid
- Distilled water: 950 mL.
- Potassium dichromate: 25 g.
- Mercuric chloride: 50 g.
- Glacial acetic acid: 50 g.
- Fixation time: 4–24 hours followed by prolonged wash.
Helly's Fluid
- Mercuric chloride: 50 g
- Potassium dichromate: 25 g
- Sodium sulfate: 10 g
- Distilled water: 950 mL.
- Fixation time: Same as in Zenker's fluid but 50 mL of 40% formaldehyde is added to this fluid before use.
PICRIC ACID FIXATIVES
It is used as a mixture and common mixtures are Bouin's fluid, Rossman's fluid and Brasil's alcoholic picro-formol fixative. It reacts with histones and basic proteins to form crystalline picrates with amino acids. This is a very good preservative for glycogen (Table 5).
Bouin's Fluid
- Saturated aqueous picric acid solution: 75 mL.
- Formalin (40% formaldehyde): 25 mL.
- Glacial acetic acid: 5 mL.
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- Picric acid (50% water): 80 g
- Formalin: 2,040 mL
- Ethanol or isopropyl alcohol: 6,000 mL.
- Trichloroacetic acid: 65 g.
FIXATION OF TISSUES FOR ELECTRON MICROSCOPY
Fixation for electron microscopy should be done at 4°C in the refrigerator. The commonly used fixatives are glutaraldehyde and osmium tetroxide. Glutaraldehyde forms cross-linkages between molecules and preserves the cellular structure well. Whereas osmium tetroxide gels protein by formation of bridges between molecules. It rapidly fixes the tissue as well as stains the tissue structures.
FACTORS INVOLVED IN TISSUE FIXATION
- Temperature: Usually done at room temperature. For electron microscopy and some histochemistry low temperature (0–4°C) is preferred to slow down the autolysis. For fixation in bacteriology (e.g. leprosy, TB) and blood film heat may be used. For urgent biopsy, formalin may be heated up to 60°C. Fixation can be done within 5 hours at 40°C but higher temperature deteriorates some antigens, e.g. PCNA.
- pH (hydrogen ion concentration): Good fixation is achieved at a pH of 6–8. Outside this pH there may be damage to the ultrastructure. Storage granules of adrenaline and noradrenaline are most stable at pH 6.5 with formalin fixative. Gastric mucosa is best fixed at pH 5.5.
- Duration: Primary fixation in buffered formalin for 2–8 hours (< 24 hours). Prolonged fixation causes hardening and shrinkage of tissue.
- Tissue penetration: This process is usually slow with usual fixatives. So, the blocks should be either thin or small (e.g. 1 mm3 for electron microscopy). The depth of penetration is proportional to the square root of time (t) and can be expressed as d = K√t; where K (in tissue) is the constant and it is the coefficient of diffusibility of the fixative in tissues or gel. K value (tissue) is high (1.33) in potassium dichromate fixative whereas it is low (0.25) in chromium and glutaraldehyde fixative.
- Concentration of fixative: Ideal concentration should be used for good fixation, e.g. 10% buffered formalin, 3% glutaraldehyde or saturated solution of picric acid and mercuric chloride. The concentration may be changed with change of pH or addition of buffer to a fixative. Like glutaraldehyde can be used at 0.25% if the pH of the vehicle is correct and can be used for immuno-electron microscopy.
- Osmolality: The preferred osmolality is slightly hypertonic solution or isotonic solution.
- Substances added as vehicles: Sodium chloride added to mercuric chloride fixative to increase the binding of it to amino groups of proteins. Likewise, tannic acid enhances fixation of protein, lipid and complex carbohydrates especially for electron microscopy.
- Volume changes: Volume of tissue may be changed during fixation. Nucleuses in frozen sections are usually bigger whereas prolonged fixation in formalin causes shrinkage. Some intercellular material like collagen swells when they are fixed.
SECONDARY FIXATION (POST-FIXATION/POST-CHROMATIN)
It is the use of two fixatives in succession. The first one is the primary fixative and the second one is the secondary fixative. As for example, after primary fixation in buffered formalin, tissues are kept in secondary fixative of mercuric chloride-formaldehyde solution. Advantage of this sublimate post-fixation is that tissues are more easily cut and flatten better, also they give better staining quality. Likewise, tissues fixed with glutaraldehyde may be post-fixed with osmium tetroxide which makes the membranes relatively permeable and better stained (Table 6).
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