19 February 2019

Fixation

  
Fixation is a procedure adopted to kill, harden and preserve materials for microscopic study, by means of a substance known as fixative. The shape, structure, intercellular relationship and chemical constituents of tissues are preserved by preventing degeneration, putrefaction, decomposition and distortion of tissues after death, consequent to cutting and removal from the body.

Effects of fixative in general:

1.  They harden soft and friable tissues and make the handling and cutting of sections easier. This is usually accelerated by the action of alcohol during the dehydration process.

2.  They make the cells resistant to damage and distortion caused by hypotonic and hypertonic solution used during tissue processing.

3.  They inhibit bacterial decomposition

4.  They increase the optical differentiation of cells and tissue components thereby rendering them more readily visible during examination

5.  They act as mordants or accentuators to promote and hasten staining, or they may inhibit certain dyes in favor of another (e.g. formaldehyde intensifies while osmium tetroxide inhibits hematoxylin).

6.  They reduce the risk of infection during handling and actual processing of tissues.

Characteristics of a good fixative:

1.  It must be cheap.

2.  It must be stable.

3.  It must be safe to handle.

4.  It must kill the cell thereby producing minimum distortion of cell constituents.

5.  It must inhibit bacterial decomposition and autolysis.

6.  It must produce minimum shrinkage of tissue.

7.  It must permit rapid and even penetration of tissues.

8.  It must harden tissues thereby making the cutting of sections easier.

9.  It must be isotonic, causing minimal physical and chemical alteration of the cells and their constituents.

This is not, however, a strict rule since there are some hypotonic solutions (i.e. Glacial Acetic Acid) producing tissue swelling, which are being used in conjunction with hypertonic solutions (e.g. Picric Acid) causing shrinkage of cells, to produce a compound which would give an optimal effect on the tissue structure.

10. It must make the cellular components insoluble to hypotonic solutions and render them insensitive to subsequent processing.

11. It must permit the subsequent application of many staining procedures to facilitate easier and more profitable examination.


Types of Fixatives:

I.     According to composition

A.  Simple fixatives – made up of only one component susbstance

1.  Aldehydes

Aldehyde fixatives are satisfactory for routine paraffin sections, for electron microscopy and when histochemical and enzyme studies are indicated.

a.  Formaldehyde (Formalin)

This is a gas produced by the oxidation of methyl alcohol, and is soluble in water to the extent of 37 – 40% weight in volume. Pure stock solution of 40% formalin is unsatisfactory for routine fixation since high formaldehyde concentrates tend to over harden the outer layer of the tissue and affect staining adversely. Therefore, it must be diluted 1:10 or 1:20 to make a 10% or 5% solution, or combined with another fixative to form a compound solution. Usual fixation time is 24 hours.

Advantages:

(1)  It is cheap, readily available, easy to prepare, and relatively stable, especially if stored in buffered solution.

(2)  It is compatible with many stains, and therefore can be used depending upon the need of the tissues.

(3)  It does not over harden tissues, even with prolonged periods of fixation, as long as solutions are regularly changed.

(4)  It penetrates tissues well.

(5)  It preserves fat and mucin, making them resistant to subsequent treatment with fat solvents, thereby allowing tissue enzymes to be studied.

(6)  It preserves glycogen.

(7)  It preserves but does not precipitate proteins, thereby, allowing tissue enzymes to be studied.

(8)  It does not make tissues brittle, and is therefore recommend for nervous tissue preservation.

(9)  It allows natural tissue colors to be restored after fixation by immersing formalin – fixed tissues in 70% alcohol for one hour, and is therefore recommended for colored tissue photographies.

(10) It allows frozen sections to be prepared easily.

(11) It does not require washing out, unless tissues have stayed in formalin for excessively long period of time.

Disadvantages:

(1) Fumes are irritating to the nose and eyes may cause sinusitis, allergic rhinitis or excessive lacrimation.

(2) The solution is irritating to the skin and may cause allergic dermatitis on prolonged contact.

(3) It may produce considerable shrinkage of tissues.

(4) It is a soft fixative and does not harden some cytoplasmic structures adequately enough for paraffin embedding.

(5) If unbuffered:

(a) Formalin reduces both basophilic and eosinophilic staining of cells, thereby reducing the quality of routine cytologic staining. Acidity of formic acid may, however, be used to an advantage when applying the Silver Impregnation Technique of staining.

(b) It forms abundant brown pigment granules on blood–containing tissues.

e.g. spleen, due to blackening of hemoglobin

(6) Prolonged fixation may produce:

(a) Bleaching of the specimen and loss of natural tissue colors.

(b) Dispersal of fate from the tissues into the fluid.

Precaution:

(1) Prolonged storage of formaldehyde especially at very low temperature, may induce precipitation of white formaldehyde deposits and produce turbidity although this, in itself, does not impair the fixing property of the solution. Precipitates may be removed by filtration or by addition of 10% methanol.

Methanol added as a preservative to formaldehyde will prevent its decomposition to formic acid or precipitation to paraformaldehyde, but it serves to denature protein, thereby rendering formalin unsuitable as a fixative for electron microscopy.

(2) Room should be properly ventilated with adequate windows and preferably with an exhaust fan to prevent inhalation of fumes and consequent injury to the eyes and nose.

(3) Dermatitis may be avoided by the use of rubber gloves when handling specimens fixed in formalin.

(4) Bleaching of tissues may be prevented by changing the fluid fixative every three months.

(5) Natural tissue color may be restored by immersing tissues in 70% alcohol after fixation.

(6) If fatty tissues are to be stored for a long time, cadmium or cobalt salts are added to prevent dispersion of fat out into the fluid.

(7) Acid reaction due to formic acid formation can be buffered or neutralized by adding magnesium carbonate or calcium carbonate to 10 – 15% formalin. This should be done on a wide mouth bottle to prevent violent explosion due to insufficient gas space for CO2 release.

(8) To improve staining and produce firmer and harder consistency, tissues fixed in formalin for 1 – 2 hours may be placed again in Helley’s fluid for 4 – 6 hours or in formol–sublimate for 4 – 16 hours (secondary fixation).

(9) If post–fixed in osmic acid, the tissue must be washed in demineralized water to prevent hypotonicity and bleaching.

b.  Glutaraldehyde – is made up of two formaldehyde residues, linked by three carbon chains, utilized for routine light microscopic work and also satisfactory for electron microscopy.

A 2.5% solution is used for small tissue fragments and needle biopsies fixed in 2 – 4 hours at room temperature. A 4% solution is recommended for larger tissues less than 4 mm thick, fixed in 6 – 8 hours up to 24 hours.

Advantages of glutaraldehyde over formalin:

(1) It has a more stable effect on tissues, giving firmer texture with better tissue sections, especially of central nervous tissues.

(2) It preserves plasma proteins better.

(3) It produces less tissue shrinkage.

(4) It preserves cellular structures better, hence, is recommended for enzyme histochemistry and electron microscopy.

(5) It is more pleasant and less irritating to the nose.

(6) It does not cause dermatitis.

Disadvantages:

(1) It is more expensive

(2) It is less stable

(3) It penetrates tissues more slowly.

(4) It reduces PAS positivity of reactive mucin. This may be prevented by immersing glutaraldehyde – fixed tissues in a mixture of concentrated glacial acetic acid and aniline oil.

2.  Metallic fixatives

a. Mercuric chloride – is the most common fixative, used in saturated aqueous solutions of 5 – 7%; it is included in many compound fixatives.

Advantages:

(1) It penetrates and hardens tissues rapidly and well.

(2) Nuclear components are shown in fine detail.

(3) It precipitates all proteins.

(4) It has a greater affinity to acid dyes and is preferred in lieu of formaldehyde for cytoplasmic staining.

(5) Trichome staining is excellent.

(6) It is the routine fixative of choice for preservation of cell detail in tissue photography.

(7) It permits brilliant metachromatic staining of cells.

(8) It is recommended for renal tissues, fibrin, connective tissues and muscles.

Disadvantages:

(1) It causes marked shrinkage of cells (this may be counteracted by addition of acid)

(2) It rapidly hardens the outer layer of the tissue with incomplete fixation of the center, therefore, thin sections should be made.

(3) Penetration beyond the first 2 – 3 millimeters is slow, hence, not more than 5 mm thickness of tissues should be used.

(4) If left in fixative for more than 1 – 2 days, the tissue becomes unduly hard and brittle.

(5) It prevents adequate freezing of fatty tissues and makes cutting of frozen tissues difficult.

(6) It causes considerable lysis of red blood cells and removes much iron from hemosiderin.

(7) It is inert to fats and lipids.

(8) It leads to the formation of black granular deposits in the tissues.

(9) It reduces the amount of demonstrable glycogen.

(10) Compound solutions containing Mercuric chloride deteriorate rapidly upon addition of Glacial Acetic Acid to Formalin

(11) It is extremely corrosive to metals.

Precaution

(1) Black deposits may be removed by adding saturated iodine solution in 96% alcohol, the iodine being decolorized with absolute alcohol in the subsequent stages of dehydration.

(2) Compound solutions must always be freshly prepared.

(3) The use of metals caps to cover the bottles containing fixative should be avoided.

(4) Contact of mercuric fixatives with personal jewelry should be avoided.

Just before use, Glacial Acetic Acid may be added to form Zenker’s solution or strong Formaldehyde may be added to form Zenker – Formol solution (Helly’s).

b. Chromate fixative

(1) Potassium dichromate – is used in 3% aqueous solution.

(a) It fixes but does not precipitate cytoplasmic structures.

(b) It preserves lipids.

(c) It preserves mitochondria (if used in pH 4.5 – 5.2, mitochondria are fixed; if solution becomes acidified, cytoplasm, chromatin bodies and chromosomes are fixed but mitochondria are destroyed.)

(2) Chromic acid – is used in 1 – 2% aqueous solution, usually as a constituent of a compound fixative. It precipitates all proteins and adequately preserves carbohydrates. It is a strong oxidizing agent; hence, a strong reducing agent (e.g. formaldehyde) must be added to chrome–staining fixative before use in order to prevent counteracting effects and consequent decomposition of solution upon prolonged standing.

c. Lead fixatives

Lead fixatives are used in 4% aqueous solution of basic lead acetate

Advantage:

(1) It is recommended for acid mucopolysaccharide.

(2) It fixes connective tissue mucin

Disadvantage:

It takes up CO2 to form insoluble lead carbonate especially on prolonged standing. This may be removed by filtration or by adding acetic acid drop by drop to lower the pH and dissolve the residue.

3.  Picric acid

Picric acid is normally used in strong saturated aqueous solution (approximately 1%)

Advantages:

a.  It is an excellent fixative for glycogen demonstration

b.  It penetrates tissues well and fixes small tissues rapidly

c.   The yellow stain taken in by tissue prevents small fragments from being overlooked

d.  It allows brilliant staining with the Trichome method

e.  It is suitable for aniline stains (Mallory’s Heidenhain’s or Mason’s method

f.   It precipitates all proteins

g.  It is stable

Disadvantages:

a.  It causes RBC hemolysis and reduces the amount of demonstrable ferric iron in tissues.

b.  It is not suitable for frozen sections because it causes frozen sections to crumble when cut.

c.   Prolonged fixation makes tissues hard, brittle and difficult to section. Tissues should not be allowed to remain in the fluid for more than 12 – 24 hours (depending on size).

d.  Picrates are formed upon protein; precipitates are soluble in water, hence, tissues must be first rendered insoluble by direct immersion in 70% ethyl alcohol.

Picric acid fixed tissues must never be washed in water before dehydration.

e.  Picric acid will produce excessive staining of tissues. To remove the yellow color, tissues may be placed in 70% ethyl alcohol followed by 5% sodium thiosulfate and then washed in running water.

f.   Picric acid is highly explosive when dry, and therefore must be kept moist with distilled water or saturated alcohol at 0.5 to 1% concentration during storage.

g.  It alters and dissolves lipids.

h.  It interferes with azure eosin method of staining; hence, tissues should be thoroughly washed with alcohol.

4.  Acetic acid

Acetic acid is normally used in conjunction with other fixatives to form a compound solution. It solidifies at 17oC, hence, the name Glacial Acetic Acid.

Advantages:

a.  It fixes and precipitates nucleoproteins.

b.  It precipitates chromosomes and chromatin materials; hence, is very useful in the study of nuclear components of the cell. In fact, it is an essential constituent of most compound nuclear fixatives.

c.   It causes tissues (especially those containing collagen) to swell. This property is used in certain compound fixatives to counteract the shrinkage produced by other components (e.g. mercury)

Disadvantages:

a.  When combined with potassium dichromate, the lipid – fixing property of the latter is destroyed (e.g. Zenker’s fluid)

b.  It is contraindicated for cytoplasmic fixation since it destroys mitochondria and Golgi elements of cells.

5.  Acetone

Used at ice cold temperature ranging from –5oC to 4oC.

Advantages:

a.  It is recommended for the study of water diffusible enzymes especially phosphatases and lipases.

b.  It is used in fixing brain tissues of diagnosis of rabies.

c.   It is used as a solvent for certain metallic salt to be used in freeze substitution technique for tissue blocks.

Disadvantages:

a.  It produces inevitable shrinkage and distortion.

b.  It dissolves fat.

c.   It preserves glycogen poorly.

d.  It evaporates rapidly.


6.  Alcohol

Alcohol rapidly denatures and precipitates proteins by destroying hydrogen and other bonds. It must be used in concentrations ranging from 70 to 100% because less concentrated solutions will produce lysis of cells. 

Advantages:

a.  It is ideal for small tissue fragments.

b.  It may be used both as a fixative and dehydrating agent.

c.  It is excellent for glycogen preservation.

d. It preserves nuclear stains.

Disadvantages:

a.  It causes RBC hemolysis

b.  It dissolves fats and lipids; as a general rule, alcohol containing fixatives are contraindicated when lipids are to be studied.

7.  Osmium tetroxide

This is a pale yellow powder which dissolves in water (up to 6% at 20oC) to form a strong oxidizing solution.

Advantages:

a.  It fixes conjugated fats and lipids permanently by making them insoluble during subsequent treatment with alcohol and xylene.

Fats from hydrated osmium dioxide, are stained black and therefore are easier to identify.

b.  It preserves cytoplasmic structures well, e.g. Golgi bodies and mitochondria.

c.   It fixes myelin and peripheral nerves well, hence, is used extensively for neurological tissues.

d.  It produces brilliant nuclear staining with safranin

e.  It adequately fixes materials for ultrathin sectioning in electron microscopy, since it rapidly fixes small pieces of tissues and aids in their staining.

f.   It precipitates and gel protein.

g.  It shows uniformly granular nuclei with clear cytoplasmic background.

h.  Some tissues (e.g. adrenal glands) are better fixed in vapor form of osmium tetroxide. This eliminates “washing out” of the fixed tissue.

Disadvantages:

a.  It is very expensive.

b.  It is a poor penetrating agent, suitable only for small pieces of tissues.

c.  It is readily reduced by contact with organic matter and exposure to sunlight, forming a black precipitate, which settles at the bottom of the container.

d. Prolonged exposure to acid vapor can irritate the eye, producing conjunctivitis, or cause the deposition of black osmic oxide in the cornea, producing blindness.

e.  It inhibits hematoxylin and makes counterstaining difficult.

f.   It is extremely volatile.

Precaution:

a.  It should be kept in a dark–colored, chemically clean bottle to prevent evaporation and reduction by sunlight or organic matter.

b.  Eyes and skin may be protected by using a fume hood or wearing protective plastic masks or gloves while using osmium tetroxide.

c.   Addition of saturated aqueous mercuric chloride solution (0.5 to 1ml / 100 ml of stock solution) will prevent its reduction with formation of black deposits.

d.  Black osmic oxide crystals may be dissolved in cold water.

e.  Osmic acid – fixed tissues must be washed in running water for at least 24 hours to prevent formation of artifacts.

f.   To prevent contact of tissues with black precipitate formed in the bottom of the jar, the tissue may be wrapped in cotton gauze and suspended in the fluid by means of a thread.

8.  Heat

This procedure involves thermal coagulation of tissue proteins for rapid diagnosis, usually employed for frozen tissue sections and preparation of bacteriologic smears.

Advantages:

a.  Fixation is better

b.  It preserves nuclear and cytoplasmic detail

c.   It is suitable for frozen tissue preparation.

Disadvantages:

a.  It produces considerable tissue shrinkage and distortion

b.  It destroys RBC

c.   It dissolves starch and glycogen.

B. Compound fixatives – are made up of two or more fixatives which have been added together to obtain the optimal combined effect of their individual actions upon the cells and tissue constituents.

II.    According to action

A. Microanatomical fixatives

Are those which permit the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of the tissues in question.

1.  10% formol saline – is a simple microanatomical fixative made up of saturated formaldehyde (40% grams by weight volume) diluted to 10% with sodium chloride; it is recommended for fixation of central nervous tissues and general post–mortem tissues for histochemical examination.

Fixation time:      24 hours at 35oC
48 hours at 20 – 25oC

Advantages:

a. It penetrates and fixes tissue evenly.

b. It preserves microanatomic and cytologic details with minimum shrinkage and distortion.

c. Large specimen may be fixed for a long time provided that the solution is changed every three months.

d. It preserves enzymes and nucleoproteins.

e. It demonstrates fats and mucin.

f.  It does not over harden tissue, thereby facilitating dissection of the specimen.

g. It is ideal for most staining techniques, including Silver Impregnation.

h. It allows natural tissue color to be restored upon immersion in 70% alcohol.

Disadvantages:

Similar to formaldehyde with the following addition:

a.  It is a slow fixative, period of fixation is required to be 24 hours or longer.

b.  Formol–saline fixed tissues tend to shrink during alcohol dehydration; this may be reduced by secondary fixation.

c.   Metachromatic reaction of amyloid is reduced.

d.  Acid dye stains less brightly than when fixed with mercuric chloride

2.  10% Neutral Buffered Formalin – is recommended for preservation and storage of surgical, post–mortem and research specimens.

Fixation time:           4 – 24 hours

Advantages:

Are similar to formol–saline with the following addition:

a.  It prevents precipitation of acid formalin pigments on post–mortem tissues.

b.  It is the best fixative for tissues containing iron pigments and for elastic fiber which do not stain well after Susa, Zenker or chromate fixation.

Disadvantages:

a.  It is longer to prepare; hence, is time consuming

b.  Positivity of mucin to PAS reduced.

c.   It may produce gradual loss in basophilic staining.

d.  Reactivity of myelin to Weigert’s Iron Hematoxylin Stain is reduced.

e.  It is inert towards lipids, especially neutral fats and phospholipids.

3.  Heidenhain’s Susa – recommended mainly for tumor biopsies especially of the skin; it is an excellent cytologic fixative.

Fixation time:                       3 – 12 hours

Advantages:

a.  It penetrates and fixes tissues rapidly and evenly.

b.  It produces minimum shrinkage and hardening of tissues due to the counter balance of the swelling effects of acids and the shrinkage effect of mercury.

c.   It permits most staining procedures to be done, including Silver Impregnation, producing brilliant result with sharp nuclear and cytoplasmic details.

d.  It permits easier sectioning of large blocks of fibrous connective tissues.

e.  Susa–fixed tissues may be transferred directly to 95% alcohol or absolute alcohol, thereby reducing processing time.

Disadvantages:

a.  Prolonged fixation of thick materials may produce considerable shrinkage, hardening and bleaching; hence, tissues should not be more than 1 cm thick.

b.  RBC preservation is poor.

c.   Some cytoplasmic granules are dissolved.

d.  Mercuric chloride deposits tend to form on tissues; these may be removed by immersion of tissues in alcohol iodine solution.

e.  Weigert’s method of staining elastic fibers is not possible in susa–fixed tissues.

Precaution:

a.  After using Heidenhain’s Susa fixative, the tissue should be transferred directly to a high grade alcohol, e.g. 96% or Absolute Alcohol, to avoid undue swelling of tissues caused by treatment with low–grade alcohol or water.

4.  Formol Sublimate (Formol Corrosive) – formol–mercuric chloride solution is recommended for routine post–mortem tissues.

Fixation time:           3 – 24 hours

Advantages:

a.  It penetrates small pieces of tissues rapidly

b.  It produces minimum shrinkage and hardening

c.  It is excellent for many staining procedures including Silver Reticulum methods.

d. It brightens cytoplasmic and metachromatic stains better than with formalin alone.

e.  Cytological structures and blood cells are well–preserved.

f.   There is no need for “washing–out”; tissues can be transferred directly from fixative to alcohol.

g.  It fixes lipids, especially neutral fats and phospholipids

Disadvantages:

a.  Penetration is slow; hence, tissue sections should not be more than 1cm thick.

b.  It forms mercuric chloride deposits.

c.   It does not allow frozen tissue sections to be made.

d.  It inhibits the determination of the extent of tissue decalcification.

5.  Zenker’s solution – is made up of Mercuric Chloride stock solution to which Glacial Acetic acid has been added just before its use. It is recommended for fixing small pieces of liver, spleen, connective tissue fibers and nuclei.

Fixation time:                       12 – 24 hours

Advantages:

a.  It produces a fairly rapid and even fixation of tissues.

b.  Stock solutions keep well without disintegration.

c.   It is recommended for Trichome staining.

d.  It permits brilliant staining of nuclear and connective tissue fibers.

e.  It is compatible with most stains.

f.   It may act as a mordant to make certain special staining reactions possible.

Disadvantage:

a.  Penetration is poor.

b.  It is not stable after addition of Acetic acid.

c.   Prolonged fixation (for more than 24 hours) will make tissues brittle and hard.

d.  It causes lysis of red blood cells and removes iron from hemosiderin.

e.  It does not permit cutting of frozen sections.

f.   It has the tendency to form mercuric pigment deposits or precipitates.

g.  Insufficient washing may inhibit or interfere with good cellular staining.

Precaution:

a.  Do not let tissues stay in solution for more than 24 hours.

b.  Solutions must always be freshly prepared.

c.   Tissues must be washed out thoroughly in running water to permit good staining.

d.  Mercuric deposits may be removed by immersing tissues in alcoholic iodine solution.

e.  Tissues should be cut thin (2 – 3 mm) and hollow organs should be opened to promote complete penetration and fixation.

6.  Zenker–Formol (Helly’s solution)

Fixation time:                       12 – 24 hours

Advantages:

a.  It is an excellent microanatomic fixative for pituitary gland, bone marrow and blood containing organs such as spleen and liver.

b.  It penetrates and fixes and tissues well.

c.   Nuclear fixation and staining is better than Zenker’s.

d.  It preserves cytoplasmic granules well.

Disadvantages:

The disadvantages of Helly’s solution are similar to Zenker’s except that brown pigments are produced if tissues (especially blood–containing organs) are allowed to stay in the fixative for more than 24 hours due to RBC lysis. This may be removed by immersing the tissue in saturated alcoholic picric acid or sodium hydroxide.

7.  Bouin’s solution is recommended for fixation of embryos

Fixation time:                       6 – 24 hours

Advantages:

a.  It produces minimal distortion of micro–anatomical structures.

b.  Yellow stain is useful when handling fragmentary biopsies.

c.   It permits brilliant staining of tissues.

d.  It preserves glycogen.

e.  It does not need “washing out.”

Disadvantages:

a.  It penetrates poorly; hence, its use is limited to small fragments of tissue.

b.  Picrates are soluble in water, hence, tissues should not be washed with running water but rather, transferred directly from fixative to 70% alcohol.

c.   It is not suitable for fixing kidney structures.

d.  It destroys cytoplasmic structures, e.g. mitochondria

e.  It produces RBC hemolysis and remove demonstrable ferric iron from blood pigments.

f.   It reduces or abolishes Feulgen reaction due to hydrolysis of nucleoproteins.

8.  Brasil’s solution

Advantages:

a.  It is better and less “messy” than Bouin’s solution.

b.  It is an excellent fixative for glycogen.

Overnight tissue fixation by automatic processing techniques may utilize 3 – 4 changes of Brasil’s fixative at 1 ½ to 2 hours each, succeeded directly by absolute alcohol.

B.  Cytological fixatives

1. Nuclear fixatives

a.  Flemming’s fluid is the most common Chrome–Osmium Acetic Acid fixative used, recommended for nuclear preparation of such sections.

Fixation time:     24 – 48 hours

Advantages:

(1) It is an excellent fixative for nuclear structures, e.g. chromosomes

(2) It permanently fixes fat

(3) Relatively less amount of solution is required for fixation (less than 10 times the volume of the tissues to be fixed)

Disadvantages:

(1) It is a poor penetrating agent; hence, is applicable only to small pieces of tissues.

(2) The solution deteriorates rapidly and must be prepared immediately before use.

(3) Chromic–Osmic acid combinations depress the staining power of hematoxylin (especially Ehrlich’s hematoxylin)

(4) It has a tendency to form artifact pigments; these may be removed by washing the fixed tissue in running tap water for 24 hours before dehydration.

(5) It is very expensive.

b.  Carnoy’s fluid – recommended for fixing chromosomes, lymph glands and urgent biopsies.

Fixation time:     1 – 3 hours

Advantages:

(1) It is considered to be the most rapid fixative.

(2) It fixes and dehydrates at the same time.

(3) It permits good nuclear staining and differentiation.

(4) It preserves Nissl granules and cytoplasmic granules well.

(5) It preserves nucleoproteins and nucleic acid.

(6) It is an excellent fixative for glycogen since aqueous solutions are avoided.

(7) It is very suitable for small tissue fragments such as curettage and biopsy materials.

(8) Following fixation, tissues may be transferred directly to absolute alcohol, thereby shortening processing time.

Disadvantages:

(1) It produces RBC hemolysis.

(2) It causes considerable tissue shrinkage.

(3) It is suitable for small pieces of tissues due to slow penetration.

(4) It tends to harden tissues excessively and distorts tissue morphology.

(5) It dissolves fat, lipids and myelin.

(6) It leads to polarization unless very cold temperature (–70oC) are used.

c.   Bouin’s fluid (see discussions above)

d.  Newcomer’s fluid

Fixation time:     12 – 18 hours at 3oC

Advantages:

(1) It is recommended for fixing mucopolysaccharides and nuclear proteins.

(2) It produces better reaction in Feulgen stain than Carnoy’s fluid.

(3) It acts both as nuclear and histochemical fixation.

2.  Cytoplasmic fixatives

a.  Fleming’s fluid without Acetic acid – made up only of chromic and osmic acid, recommend for cytoplasmic structures particularly the mitochondria. The removal of acetic acid from the formula serves to improve the cytoplasmic detail of the cell.

Fixation time:     24 – 48 hours

Advantages and disadvantages is the same as Fleming’s solution.

b.  Helly’s fluid

Fixation time:     12 – 24 hours

Advantages:

(1) It is an excellent microanatomic fixative for pituitary gland, bone marrow and blood containing organs such as spleen and liver.

(2) It penetrates and fixes tissues well.

(3) Nuclear fixation and staining is better than Zenker.

(4) It preserves cytoplasmic granules.

Disadvantages:

The disadvantages of Helly’s solution are similar to Zenker’s except that brown pigments are produced if tissues (especially blood containing organs) are allowed to stay in the fixative for more than 24 hours due to RBC lysis. This may be removed by immersing the tissue in saturated alcoholic Picric acid or Sodium hydroxide.

c.  Formalin with “post–chroming”

d. Regaud’s fluid (Moller’s fluid)

Fixation time:     12 – 48 hours

Advantages:

(1) It penetrates tissues well.

(2) It hardens tissues better and more rapidly than Orth’s fluid.

(3) It is recommended for demonstration of chromatin, mitochondria, mitotic figures, Golgi bodies, RBC and colloid– containing tissues.

Disadvantages:

(1) It deteriorates and darkens on standing due to acidity; hence, the solution must always be freshly prepared.

(2) Penetration is slow, hence, tissues should not be thicker than 2 – 3 mm.

(3) Chromate–fixed tissues tend to produce precipitates of sub–oxide, hence should be thoroughly washed in running water prior to dehydration.

(4) Prolonged fixation blackens tissue pigments, e.g., melanin; this may be removed by washing the tissue in running tap water prior to dehydration.

(5) Glycogen penetration is poor; it is therefore, generally contraindicated for carbohydrates.

(6) Nuclear staining is poor.

(7) It does not preserve fats.

(8) It preserves hemosiderin less than buffered formalin.

(9) Intensity of PAS reaction is reduced.

e.  Orth’s fluid

Fixation time:     36 – 72 hours

Advantages:

(1) It is recommended for study of early degenerative process and tissue necrosis.

(2) It demonstrates rickettsia and other bacteria

(3) It preserves myelin better than buffered formalin.

Disadvantages:

Same as Regaud’s fluid

C. Histochemical fixative

1.  Formol saline 10%

This is a simple microanatomical fixative made up of Standard Formaldehyde diluted to 10% with sodium chloride; it is recommended for fixation of Central Nervous Tissues and general post–mortem tissues for histochemical examinations.

Fixation time:      24 hours at 35oC;

48 hours at 20 – 25oC

2.  Absolute Ethyl alcohol

3.  Acetone – used at ice cold temperature ranging from –5oC to 4oC

Advantages:

a.  It is recommended for the study of water diffusible enzymes especially phosphatases and lipases.

b.  It is used in fixing brain tissues for diagnosis of rabies.

c.   It is used as a solvent for certain metallic salts to be used in freeze substitution techniques for tissue blocks.

Disadvantages:

a.  It produces inevitable shrinkage and distortion.

b.  It dissolves fat.

c.   It preserves glycogen poorly.

d.  It evaporates rapidly. 

4.  Newcomer’s fluid

Fixation time:                       12 – 18 hours at 3oC

Advantages:

a.  It is recommended for fixing mucopolysaccharides and nuclear proteins

b.  It produces better reaction in Feulgen stain than Carnoy’s fluid

c.   It acts both as a nuclear and histochemical fixative


Related fixation term:

1.  Secondary fixation – is the process of placing an already fixed tissue in a second fixative in order to:

a.  To improve the demonstration of particular substance

b.  To make special staining techniques possible (with secondary fixative reacting as mordant).

c.   To ensure further and complete hardening and preservation of tissues.

This maybe done before dehydration and on deparaffinized sections before staining, usually with 10% formalin or 10% formol saline as a primary fixative.

2.  Post–chromatization – secondary fixation whereby a primarily fixed tissue is placed in aqueous solution of 2.5 – 3% potassium dichromate for 24 hours, to act as a mordant for better staining effects and to aid in cytologic preservation of tissues.

3.  Washing out – is the process of removing excess fixative from the tissue after fixation in order to improve staining and remove artifacts from the tissues. Several solutions may be used.

a.  Tap water – is used to remove:

(1)  Excess chromates from tissues fixed in Helly’s, Zenker’s and Fleming’s solution.

(2)  Excess formalin

(3)  Excess osmic acid

b.  50 – 70% alcohol – is used to washout excess amount of picric acid (Bouin’s solution)

c.   Alcoholic iodine – is used to remove excessive mercuric fixative.


Factors that affect fixation of tissues:

A. Retarded by

1.  Size and thickness of the tissue specimen – larger tissues require more fixatives and longer fixation time.

2.  Presence of mucus – prevents complete penetration of fixative; hence, tissues that contain mucus and fixed slowly and poorly. Excess mucus may be washed away with normal saline solution.

3.  Presence of fat – fatty tissues should be cut in thin sections and fixed longer.

4.  Presence of blood – tissues containing large amount of blood (e.g. blood vessels and spleen) should be flushed out with saline by arterial cannulization before fixing.

5.  Cold temperatures – inactivates enzymes

B. Enhanced by

1.  Size and thickness of tissues – smaller and thinner tissues require less fixative and shorter fixation time.

2.  Agitation – fixation is accelerated when automatic or mechanical tissue processing is used.

3.  Moderate heat (37 – 56oC) accelerates fixation but hastens autolytic changes and enzyme destruction.

PRINCIPLES AND PRECAUTIONS IN HANDLING AND FIXATION OF SPECIMENS IN GENERAL

1.  Autopsy materials should be fixed as soon after death as possible to prevent decomposition and autolysis due to deprived oxygen and metabolism. If this is not possible, the body should be placed in a mortuary refrigerator (kept at temperature of 4oC) or undergo arterial embalming for better tissue preservation.

2.  Surgical specimens should be fixed as soon as possible after removal or refrigerated if fixation is to be delayed, to prevent drying of surface layers and ultimate tissue distortion.

If placed in NSS during the operation, autolysis may occur before fixation is carried on.

3.  All tissue specimens must be properly labeled and identified.

4.  If tissues are refrigerated, slow freezing of unfixed tissues near 0oC must be avoided since this may promote formation of ice crystal artifacts. Repeated freezing and thawing, on the other hand, will destroy cellular organelles, release enzymes and diffuse soluble components of the cell.

5.  Tissue slices should be taken at right angles to the surface of the organ and should be sufficiently deep to show the normal anatomic components.

6.  Tissues should not be more than 5 mm thick except in lung edema (in which case tissue slices may be 1 – 2 cm thick), with minimum squeezing and handling.

Thin sections allow complete penetration by fixative in a short time.

7.  Purulent materials, exudates or transudates should be marked and kept for possible cultures, smears and other bacteriologic examinations.

8.  The amount of fixative must be adequate, approximately 20 – 50 times the volume of the tissue specimen except in osmium tetroxide which is very expensive requiring only 5 – 10 times that of tissue volume for fixation.

9.  Contamination of fixed tissue with precipitate (e.g. Osmium tetroxide), should be avoided.

10. In most instances, fixed tissues must be washed thoroughly to remove salts and/or pigments before staining.

11. Low temperature retards fixation but prevents autolysis, therefore tissues should be fixed at a temperature near the freezing point of the fixative.

12. The required period for fixation should not be exceeded since this may cause excessive hardening or brittleness of tissues.

13. There must always be an adequate supply of fixatives at all times.

14. Drying should be avoided to prevent shrinkage and distortion of tissue with loss of cellular detail.

Small tissue biopsies may be placed in a petri dish with moistened filter paper to prevent drying.

15. Solid organs should be injected with, as well as immersed in, enough fixatives to ensure complete penetration and fixation.

16. Hollow organs (e.g. stomach, intestines) should be packed with cotton soaked in fixative or completely opened before being immersed in adequate fixing solution.

17. Air–filled lungs may float on fixative. To avoid this, the organ may be covered with several layers of gauze to maintain it under surface.

18. Human brains should undergo intravascular perfusion (washing out of blood with Ringer’s lactate). This may, however, lead to artifact formation with loss of blood content. They may be suspended by a cord tied under the Circle of Willis to prevent flattening.

19. Dense tissues are poorly penetrated, hence require long fixation.

20. Eyes should not be dissected before they are fixed since this may lead to immediate tissue collapse and wrinkling due to escape of vitreous humor. They are not, however, easily penetrated due to tough sclera. Formol – alcohol must be injected before immersing the organ in the fixative.

21.  Frozen sections may lead to formation of ice crystals artifacts.

21. When fixing muscles, to avoid rigor contraction and staining of artifacts, the fresh biopsy material may be stretched for 30 minutes by sutures on each end, and left in a moist filter paper placed in a petri dish or suspended in fixative.

22. Water should not be used for glycogen–containing tissues because glycogen is soluble in water.

23. To assure rapid access of the fixative to all parts of the tissue, the tissue may be minced, that is, small pieces of specimen may be divided into small fragments (½ to 1 x 1 mm) and transferred to the vial of a fixative by means of a toothpick.

24. Hard tissues (e.g. cervix, uterine, fibroids, hyperkeratotic skin, fingernails, etc.) may be washed out in running water overnight and immersed in 4% aqueous phenol solution for 1 – 3 days (Lendrum method). This will soften the tissue and allow easier sectioning without producing any marked distortion of the cell structure.

Proper tissue processing should start with proper fixation and preservation since a badly preserved tissue will later on yield a badly processed specimen and may prove to be unsuitable for study. The choice of fixative and mode of processing must therefore vary depending upon the following factors:

1.  Need for immediate examination.

2.  Tissue structure or component to be studied.

3.  Type of tissue to be processed.

4.  Staining technique to be applied.

5.  Type of specimen to be made, whether serial or individual.

Good cutting and staining of section is greatly dependent upon proper fixation. Errors in the choice and use of fixative have to a very large extent adversely affected and caused ultimate failure in the processing of tissues under investigation.

Some of the difficulties encountered because of improper fixation are:

1.  Failure to arrest early autolysis of cells

Cause:

Failure to fix immediately (the tissues was probably allowed to dry before fixing); Insufficient fixative

2.  Removal of substances soluble in fixing agent

Cause:

Wrong choice of fixative

3.  Presence of artifact pigments on tissue sections

Cause:

Incomplete fixation

4.  Tissues are soft and feather–like in consistency

Cause:

Incomplete fixation

5.  Loss or inactivation of enzymes needed for study

Cause:

Wrong choice of fixative

6.  Shrinkage and swelling of cells and tissue structure

Cause:

Overfixation

7.  Tissue blocks are brittle and hard

Cause:

Prolonged fixation

An incompletely fixed tissue may lead to improper and incomplete clearing and impregnation and may later prove to be a hindrance to normal sectioning and staining of specimen.


HOPE: Herpes–glutamic acid buffer mediated Organic Solvent Protection Effect

The discovery of the so-called HOPE method allows tissue samples to be treated such that they do not only meet the requirements of clinical histology, but can still be characterized later on by modern methods of proteomics, a technique analyzing all proteins at once. This is successful, since the structure of the tissue is "fixed" in a way that the protein molecules remain accessible for systematic analysis. This technique therefore meets current requirements in terms of a more personalized medicine and thus opens up new opportunities for researching diseases and their therapies.

HOPE stands for "Hepes-glutamic acid buffer mediated Organic solvent Protection Effect" and is a method for preserving tissue samples for later analysis.

A look at a tissue sample through the microscope tells researchers and pathologists a whole story about a patient's health status. In order to preserve the tissue, samples are taken and usually fixed with formalin, before they are embedded in wax-like paraffin and cut into razor-thin slices. These are then stained and allow the experienced eye to discern tissue structures and make diagnoses and prognoses.

One disadvantage of this type of sample preparation is that formalin cross-links the protein molecules that are present in the cell. This makes them difficult to analyze. In order to carry out analyses of this type, snap-frozen samples are used.

Snap-freeze is a term often used in scientific papers to describe a process by which a sample is very quickly lowered to temperatures below –70°C. This is often accomplished by submerging a sample in liquid nitrogen. This prevents water from crystallizing when it forms ice, and so better preserves the structure of the sample (e.g. RNA, protein, or live cells)