Chapter 1 - Fixation

Fixative

• Alters tissue by stabilizing proteins so that it’s resistant to further changes

• Must change soluble to insoluble

• Occurs chemically (fixative solutions) or physically (heat, desiccation)

Denaturation

Causes protein to unfold and internal bonds to become disrupted

Additive Fixation

• denaturation that allows for the protein to combine with the fixative molecule which causes protein to become insoluble

Non-additive Fixation

• denaturation causes protein to become less capable of remaining in an intimate relationship with water, becoming more reactive

• Protein does not combine with fixative molecule

Old definition of fixation

Kills, penetrates, and hardens

Pentration

• very important

• Allows for the inner portion of the tissue to become fixed as well as a few exterior cellular layers

Hardening

No longer important with development of fixation technology

Fixative Functions (3)

• kills tissue

• Helps maintain proper relationship between cells and extracellular substances

• Aids in rendering cell constituents insoluble

Kills Tissue

• prevents decay/putrefaction, or autolysis

• Decay is considered a bacterial attack and is prevented by strict antiseptic techniques

• Autolysis is enzyme attack and if tissue is severely autolyzed, staining will not work; this can not be prevented

Help maintain proper relationship between cells and extracellular substances

• examples: connective fibers and amorphous ground substances

• Stabilization is very important during subsequent processing steps that might otherwise distort tissue elements

Refractive index

• ratio of visibility of light in air to light in liquid or a solid

• Fixatives also bring out differences in refractive indexes to increase visibility/contrast between different tissue elements

Functions vs Actions of Fixatives

• Functions: what their purpose is? what they do for us in IHC?

• Actions: how they do it? What do they specifically do to that histology specimen

Actions of Fixatives

• enzymes rendered inactive as result of protein-stabilizing actions of fixatives

• Fixative kill bacteria and molds which cause putrefaction

• Fixatives make tissue more susceptible to dyes, may act as mordants, which link tissue to dye

• Tissues rich in enzymes (liver, pancreas, brain) or more susceptible to rapid autolysis vs tissues with more connective fibers

Heat (physical) Actions of Fixatives

• will stabilize and denature the protein

• generally not used but due to the microwave oven it’s used more now

Desiccation (physical) Actions of Fixatives

• rarely used

• ex: air-drying TP for WG

Using one or more reagents (chemical) Actions of Fixatives

• reagents can be classified as additive/nonadditive, coagulant/noncoagulant

Additive Fixatives

• chemically link or add themselves to tissue, changing it

• electrical charge at binding site may change, if it was once a force that was helping keep the shape of the molecule, the tertiary structure may change

• ex: mercuric chloride, chromium trioxide, picric acid, formaldehyde, glutaraldehyde, osmium tetroxide, zinc sulfate, chloride

Nonadditive Fixatives

• act on tissue without chemically combining

• these fixatives dissociate bound water molecules within the tissue causing it to potentially shrink or harden if overexposure occurs

• ex: organic compounds like acetone and alcohols

Coagulant Fixatives

• establish a network in tissue that allows solutions to readily penetrate or gain entry into the interior of the tissue

• ex: alcohol, zinc salts, mercuric chloride, chromium trioxide, picric acid

Noncoagulant Fixatives

• creates a gel that makes penetration by the subsequent solution difficult

• ex: formaldehyde, glutaraldehyde, osmium tetroxide, potassium dichromate, acetic acid

Factors Influencing Fixation

• temperature

• size

• volume ratio

• time

• choice of fixative

• penetration

• tissue storage

• pH

• osmolality

Temperature

• temperature is directly related to the rate of fixation and the rate of autolysis and diffusion of cellular elements (increase in temp. = increase in rate of fixation)

• 0-4 C is ideal for fixation of specimens for electron microscopy

• elevated temperatures are being used for fixation in both tissue processors and ovens

• temperatures of up to 45 C are reported to have little effect on tissue morphology

Size

• thickness is important because of its effect on reagent preparation

• if large specimens are held for extended periods without being surgically opened to expose all layer, fixative will have difficulty penetrating, resulting in autolysis of epithelium

• sections should be no more than 3 mm thick for routine processing schedules

• sections should never be so thick that it touches the top and bottom of the cassette

Volume Ratio

• fixative volume should be at least 15-20 times greater

• staining problems are frequently the result of poor fixation due to the use of not enough fixative solution

Time

• ideally the tissue should be placed in fixative immediately after surgical removal and autopsies should be performed immediately after death

• the more time that elapses between interruption of the blood supply and fixation, the more post mortem changes that can be demonstrated microscopically

• staining problems are frequently the result of poor fixation due to the use of an inadequate volume of fixative

Choice of Fixative

• method of fixation must be chosen as soon as specimen is presented

• sometimes fixation is not required (if immunofluorescence study or enzyme profile)

Penetration

• factors that determine the minimum length of time a fixative should act are the rate of penetration and the mode of action

• formaldehyde (a noncoagulant fixative) penetrates fast, continues to cross-link proteins for a long time after penetration is complete

• most coagulant fixatives achieve full effect on tissue at any particular depth as they have penetrated to that depth at a concentration sufficient to cause coagulation

• fixatives in order of decreasing speed of penetration: formaldehyde>acetic acid>mercuric chloride>methyl alcohol>osmium tetroxide>picric acid

Tissue Storage

• storage isn’t usually a problem with tissue fixed in a neutral buffered formalin because the tissue may remain in this solution indefinitely

• appropriate storage will be described in individual sections on each fixative solution

p

pH

• not important in light microscopy; varying pH from 4 to 9 makes little difference in fine structure produced by formalin fixation

• important in electron microscopy

• when ultrastructural preservation is the main purpose of fixation, solution should be buffered to a pH of 7.2-7.4 (physiological pH)

Osmolality

• number of particles in solution

• not important in light microscopic studies as it is in ultrastructural studies

• body fluids have an osmolality about 340 mOsm

• isotonic, hypertonic, hypotonic

Isotonic

• normal saline solution used as a holding solution for tissue but not for prolonged periods of time

• cells can be placed in this solution type without shrinking or bursting

• .9% sodium chloride or 5% glucose solution

Hypertonic

• cell in a solution that’s more concentrated or contains more particles than the cell cytosol

• cell shrinks

• water leaves the cell and goes to the solution outside

Hypotonic

• cell is placed in a solution that contains fewer dissolved particles than the cell cytosol causing the swell to cell with the potential of rupturing the membrane

The Nucleus

• DNA and RNA found within

• controls and regulates all cellular activity

• most fixatives don’t chemically react with it

• acetic alcohol and Carnoy solution are preferred fixatives for nucleic acids

• formaldehyde won’t react until temp reaches 45 C for RNA and 65 for DNA

• coagulating or precipitating fixatives render tissues more resilient to disruptive effects of sectioning, deparaffinization, and staining (resulting in sharper and more intact nuclei)

• following formalin fixation nuclei can show coalescence of chromatin into strands with intervening clear spaces “Nuclear Bubbling”

Proteins

• primary: singular chain of amino acids (arrangement of their covalent bonds)

• secondary: hydrogen bonding between various components of the peptide chain (alpha helix and beta plated sheets)

• tertiary: total 3D structure

• quaternary: where the 3D subunits come together

• additive fixatives can alter the 3D shape of proteins by changing electrical charges at the site of attachment

• nonadditive, coagulant fixatives cause proteins to become insoluble by altering their tertiary structure

• methanol and ethanol preserve the secondary structure of proteins while markedly affecting their tertiary structure

Lipids

• several fixatives will preserve lipids

• osmium tetroxide and chromic acid fix lipids so that they aren’t lost in the subsequent processing steps

Carbohydrates

• some are lost during fixation

• retention of glycogen (storage form of glucose) is thought to result from entrapment by the fixed proteins

Simple Aqueous (Water Based) Fixatives or Fixative ingredients

• acetic acid

• formaldehyde

• glutaraldehyde

• mercuric chloride

• osmium tetroxide

• zinc salts

• picric acid

• potassium dichromate

• others

Acetic Acid

• concentrated = glacial AA

• noncoagulant fixative

• penetrates rapidly, leaving tissue very soft

• major use is precipitation and preservation of nucleoproteins (added to many fixative mixtures because of its ability to fix nuclei)

• swells proteins more than any other fixative (can be added to other fixatives to counteract the shrinking effect of other reagents

• doesn’t fix or destroy carbohydrates or fix lipids

• lyses RBCs

• should be stored at room temperature and away from strong oxidizers, nitric acid, and strong caustics

• can cause severe burns and has permissible exposure limit of 10 ppm (must be transported in an acid carrier and used under a hood; add acetic acid to water, not water to acetic acid)

Formaldehyde pt. 1

• commercial solutions are 37-40% formaldehyde but are considered 100% formalin

• 10% formalin is most commonly used fixative

• paraformaldehyde (highly polymeric form of formaldehyde; used in electron microscopy labs because it yields a pure formaldehyde solution)

• noncoagulant additive fixative

• reacts with tissue groups (primarily groups found in amino acids that contain a reactive hydrogen)

• greatest binding occurs between pH of 7.5 and 8

• preserves lipids but not made insoluble (gradual loss of lipids with prolonged storage in formalin)

• penetrates quickly, fixes slowly because it takes a long time to cross-link tissue proteins

• causes less shrinkage than any other fixative

• hardens tissue more than any other fixative except ethanol and acetone

Formaldehyde pt. 2

• black acetic hematin or formalin pigment

  • formaldehyde can be used in a simple aqueous solution or with addition of sodium chloride, but these solutions become acidic, frequently resulting in the formation of black acid hematin, also called Formalin Pigment

    • microcrystalline dark brown pigment

    • tend to form when pH of solution drops below 6.0

    • undesirable, may mask or stimulate microorganisms and pathologically relevant pigments

    • reduces silver solutions used in procedures for staining melanin, fungi, reticulin, and spirochetes

    • birefringent and can be monitored by polarization

• can be prevented by maintaining a neutral pH of the solution

• can be removed by treating tissue sections with alcoholic picric acid or alkaline alcohol

Formaldehyde Solutions

• formaldehyde solutions are very hypertonic, but formaldehyde molecule not osmotically active

• 10% Aqueous Formalin, 10% Formalin Saline, Calcium Formalin, Formalin Ammonium Bromide, Acetate Formalin, 10% Neutralized Formalin, 10% Neutral Buffered Formalin, Modified Millonig Formalin, Alcoholic Formalin

10% Aqueous Formalin

• 100 mL Formaldehyde (37-40%) + 900 mL distilled water

• hypotonic, may produce formalin pigment

10% Formalin Saline

• 100 mL Formaldehyde (37-40%) + 9g Sodium Chloride + 900 mL distilled water

• isotonic may produce formalin pigment

Calcium Formalin

• 100 mL Formaldehyde (37-40%) + 10g Calcium Chloride + 900 mL distilled water

• recommended for fixation of phospholipids

Formalin Ammonium Bromide

• 150 mL Formaldehyde (37-40%) + 20g Ammonium Bromide + 850 mL distilled water

• only CNS tissue, with Cajal astrocyte procedure

• very acidic, lyses RBCs, nuclei give direct positive Schiff reaction due to Feulgen hydrolysis during fixation

Acetate Formalin

• 100 mL Formaldehyde (37-40%) 20g sodium acetate + 900 mL distilled water

• one of the better formalin solutions if you don’t want to prepare the buffered reagent

10% Neutralized Formalin

• 100 mL Formaldehyde (37-40%) + Calcium or magnesium carbonate to excess + 900 mL distilled water

• not recommended; becomes acidic

10% Neutral Buffered Formalin

• 100 mL Formaldehyde (37-40%) 4g sodium phosphate monobasic + 6.5g sodium phosphate dibasic + 900 mL distilled water

• recommended for routine formalin fixation, hypotonic

Modified Millonig Formalin

• 100 mL Formaldehyde (37-40%) + 18.6g monobasic sodium phosphate + 4.2g sodium hydroxide + 900 mL distilled water

• isotonic, pH of 7.2-7.4

• dual purpose, allowing electron microscopy

Alcoholic Formalin

• 100 mL Formaldehyde (37-40%) + 650 mL absolute ethyl alcohol + 250 mL distilled water

• compound fixative, but categorized with other formalin solutions

• useful on tissue processors, because it fixes and dehydrates

• components must be measured separately, and poured individually into a flask for mixing

Formaldehyde

• a carcinogen

• employee exposure must be monitored

• permissible exposure limit for an 8 hour period currently set at .75 ppm

• short term exposure limit of 2 ppm over a 15 minute period

Glutaraldehyde

• similar to formaldehyde, but is a dialdehyde with 1 aldehyde group on each end of it’s molecule

• penetrates slowly and poorly

• most frequently used for the fixation of specimens for electron microscopy (2 to 4% concentrations)

  • preserves ultrastructure better than any of the aldehyde

  • tends to overharden tissue, so 2 hours or less is recommended for fixation, then transferred into buffer solution for holding

• left free to react in any method using Schiff reagent

  • techniques using Schiff reagent cannot be used on glutaraldehyde - fixed tissues due to false positives

• unstable substance that breaks down on exposure to oxygen

• for electron microscopy, small, sealed vials of glutaraldehyde should be used

  • commonly used in cacodylate or phosphate buffered solutions

• the phosphate buffered solution is prepared by: (must be made right before use)

  • sodium phosphate, monobasic: 1.9g

  • sodium hydroxide: .43 g

  • distilled water: 100 mL

  • mix well and add 18 mL of 25% glutaraldehyde solution

Mercuric Chloride

• very corrosive, all contact with metallic objects must be avoided

• mercury used on compound fixatives because it’s a powerful protein coagulant and enhances staining by leaving tissue receptive to dyes

  • not known to react with lipids or carbohydrates

  • presence in tissue inhibits freezing, making frozen sections difficult to prepare

  • penetrate poorly and will produce shrinkage. tissue exposed beyond recommended time hardens excessively

• also an additive fixative

  • reacts in acidic fixative solutions with the sulfhydryl groups of the amino acid cysteine to form cross-links between protein chains

  • leaves tissue more receptive to staining than any of the other fixative reagents

• fixation pigment produced by mercury is one that cannot be prevented but can be removed

  • unless pigment removed, it appears as either crystalline or amorphous brown precipitate lying on top of stained section

  • mercury pigment will polarize light

  • this extrinsic artifact is usually removed by treating the microscopic sections with iodine followed by sodium thiosulfate

• mercury is very toxic

  • fixatives containing it must be collected for appropriate disposal

  • careful records must be maintained. the amount remaining in lab plus amount discarded must equal amount received

Osmium Tetroxide

• primary use in the fixation of specimens for electron microscopy

  • fixed primary in aldehyde solution, then post-fixed primarily in osmium tetroxide to ensure preservation of the lipids

  • hazardous, vaporizes

Picric Acid

• only substance used both as a fixative ingredient and as a stain

• coagulant fixative of nucleoprotein but leaves DNA soluble

• will decalcify tissue containing small calcium deposits (ex: breast tissue)

• leaves tissue very receptive to acid dyes and gives tissue good soft consistency

• only fixative reagent allowing greater final shrinkage than picric acid is ethanol

• must be washed out of tissue: with 50% alcohol

• explosive compound: safe as long as it stays moist

Potassium Dichromate

• noncoagulant unless used in an acid solution

• will attach to some lipids making them insoluble but doesn’t preserve lipids to degree osmium tetroxide does

• preserves mitochondria

• can yield another of the fixation pigments

• should be removed by washing with running water

• highly toxic

Zinc Salts

• Zinc Sulfate (ZnSO4) can be a replacement for mercury

• not considered hazardous waste (can be disposed of in sink)

• superior nuclear detail and better paraffin infiltration are obtained with zinc formalin than with formalin alone

• zinc ions stabilize the protein macromolecules against the conformational changes and significant cross-linking induced by formaldehyde that are unfavorable to IHC

Compound/Combined Fixatives

• other than formaldehyde and glutaraldehyde, most fixative solutions are combined so that the disadvantage of one component will be counterbalanced by and advantage of another

• more commonly used compound fixatives:

  • B-5

  • Bouin

  • Gendre

  • Hollande

  • Zenker and Helly

  • Orth

  • Zamboni

  • Zinc Formalin

B-5 Fixatives

• stock solution: 12g mercuric chloride, 2.5 anhydrous sodium acetate, 200 mL distilled water

• working solution: 20 mL B-5 stock solution, 2 mL 37-40% formaldehyde

• wide acceptance as a fixative of hematopoietic and lymphoreticular tissues; because tissue fixed in this solution will demonstrate beautiful nuclear detail

• excess fixative washing not needed, but mercurial pigment must be removed (done with solution of iodine and then sodium thiosulfate)

• after fixation, wet tissue must be placed in storage solution (often 70% alcohol)

Bouin Solution

• 750 mL picric acid saturated aqueous solution (1.2%), 250 mL 37-40% formaldehyde, 50 mL acetic acid, glacial

• RBCs are lysed

• Iron and small calcium deposits usually dissolved, formalin pigment may be obtained

• excellent for tissue that is to be trichrome stained and for preserving structures with soft and delicate textures

• sections easily, yellow color must be removed by washing with 50-70% alcohol (remaining wet tissue should be store in 70-80% alcohol)

• maximum fixation time should be less than 24 hours

• excellent for use on GI tract bx specimens (nuclei are crisper and better stained than after fixation with 10% neutral buffered formalin)

• endocrine system tissue is well fixed and many antibodies react well

• cannot be used for preservation of tissue that must be examined ultrastructurally (electron microscopy)

Gendre Solution

• 800 mL 95% alcohol saturated with picric acid, 150 mL 37-40% formaldehyde, 50 mL glacial acetic acid

• alcoholic Bouin solution

• excellent for some carbohydrate preservation, especially glycogen

• remove excess picric acid by washing with 80% alcohol

Hollande Solution

• 25g Copper acetate, 40g picric acid, 100 mL Formaldehyde (37-40%), 15 mL acetic acid, 1,000 mL distilled water. Dissolve without heat

• modification of Bouin solution

• stable and will decalcify small specimens of bone

• cupric acetate present stabilizes RBC membranes and granules of eosinophils and endocrine cells, so less lysis occurs with Hollande solution than with Bouin

• Must be washed out before specimen placed in phosphate-buffered formalin solution on the tissue processor (salts will form an insoluble phosphate precipitate)

Zenker and Helly (Zenker-Formol) Solutions

• Zenker and Helly Stock Solution:

  • 50g Mercuric chloride, 25g Potassium dichromate, 10g sodium sulfate (optional), 1,000 mL distilled water

  • stable solution

• Zenker Working Solution:

  • 95 mL Zenker-Helly stock solution, 5 mL acetic acid, glacial

  • stable solution

• Helly Working Solution:

  • 95 mL Zenker-Helly stock solution, 5 mL 37-40% formaldehyde

  • not stable (formaldehyde is a reducing agent)

  • tissues must be treated for mercury pigment

• Zenker solution will lyse RBCs

  • Helly solution preserves RBCs but Zenker is better nuclear fixative due to presence of acetic acid

• With Zenker solution, recommended if the Mallor phosphotungstic acid-hematoxylin stain is to be applied

  • exception is silver staining; many silver techniques are unsatisfactory after fixation in Helly or Zenker solution

• maximum time for fixation is 24 hours

  • after fixation, washed in running water. remaining wet tissue stored in 70-80% alcohol

Orth Solution

• 2.5g potassium dichromate, 1g sodium sulfate, 100 mL distilled water, 10 mL formaldehyde (37-40%)

• not a good general purpose fixative

  • preferred fir subsequent demonstration of chromaffin granules in cytoplasm of cells of the adrenal medulla

  • granules colored orange and brown by chromate

  • granule demonstration may be important in diagnosis of pheochromocytoma

• tissue must be washed after fixation, time of fixation must be controlled, and remaining wet tissue stored in 70-80% alcohol

Zamboni Solution (Buffered Picric Acid - Formaldehyde or PAF)

• 20g Paraformaldehyde, 150 mL picric acid, saturated aqueous (double-filtered) heated to 60 C to dissociate the paraformaldehyde. Add 2.52% aqueous sodium hydroxide dropwise to alkalinize solution

• filer solution and allow to cool. Dilute solution to 1,000 mL with phosphate buffer prepared by: 3.31 g NaH2PO4H2O, 17.88g NaH2PO4 (anhydrous), and 1,000 mL distilled water

• should have final pH of 7.3

• good general purpose fixative, some institutions prefer it as a primary fixative for electron microscopy

Alcoholic Zinc Formalin

• 4.5g Zinc Chloride, 1,600 mL deionized water, stir until dissolved then add 2,000 mL 99% isopropyl alcohol, 400 mL of formaldehyde (37-40%)

• recommended as postfixative solution, following fixation with neutral buffered formalin

• antigenicity is enhanced, nuclear detail improved over formalin fixation

• fix 1.5 times faster than the aqueous solutions

Unbuffered Aqueous Zinc Formalin

• 1g of Zinc Sulfate, 900 mL deionized water, stir until dissolved and add: 100 mL formaldehyde (37-40%)

• formalin pigment (acid hematin) can be produced

• if zinc formalin is to be followed by a neutral fixative such as phosphate-buffered formalin, the tissue must be washed between reagents, to prevent precipitate

• 4-6 hour minimum fixation for bx tissues

• 6-8 hour minimum fixation for other tissues

Nonaqueous FIxatives

• nonadditive, coagulating fixatives

• very flammable and must be stored in fireproof cabinets

• only used when desired tissue components are destroyed or dissolved by the aqueous fixatives (reagents tend to overharden tissue drastically)

• acetone, alcohol, carnoy solution

Nonaqueous Fixatives - Acetone

• nonadditive, coagulating fixative historically used when demonstration of enzymes (especially acid and alkaline phosphatase) was indicated on tissue to be processed for paraffin embedding

• fixation done rapidly at refrigerator temperature

• also can be used as fixative for brain tissue when subsequent staining techniques for rabies diagnosis as needed

• frequently used on frozen sections of tissue to be stained for cell surface antigens by immunohistochemical techniques

• causes extreme shrinkage, distortion and overhardening

• narcotic in high concentrations, skin contact can lead to defatting and dermatitis

Nonaqueous Fixatives - Alcohol

• alcohol is a nonadditive protein precipitant (acts by breaking hydrogen and ionic bonds)

• methyl alcohol (used as a fixative for TP and blood smears; much more toxic than ethyl alcohol)

• ethyl alcohol:

  • used to preserve water-soluble tissue components (glycogen and urate crystals in gout)

  • preserves most pigments, dissolves fat, overhardens and shrinks tissue

  • alcoholic formalin fixes tissue, begins dehydration, preserves glycogen well, and penetrates quickly. when combined with formalin, the shrinkage effect of alcohol is minimized

Nonaqueous Fixatives - Carnoy Solution

• absolute ethyl alcohol: 60 mL, Chloroform: 30 mL, Acetic acid/glacial: 10 mL

• RBC’s are lysed, sometimes used in cytology for this purpose

• rapid acting, preserves glycogen, exhibits good nuclear preservation

• causes excessive shrinkage and hardening (fixation should not be prolonged beyond 4 hours)

• should only be used as indicated for the preservation of special tissue components lost through routine fixation

  • tissues should be processed through 95% alcohol, absolute alcohol, and xylene as usual, or the processing procedure can be started with absolute alcohol, if desired

Transport Solutions

• if unfixed tissue is to be held for only a brief period:

  • place it on saline dampened gauze, enclose in tightly closed plastic container, then placed in ice

• if unfixed tissue is to be held for several days:

  • Michel transport medium

  • important to maintain pH of transport medium at 7.0-7.2 because lower pH can cause variable results

Removal of Fixation Pigments

• Formalin pigment resists extraction by most strong acids, water, alcohol, or acetone

• before staining with any desired technique, both formalin and malarial pigments may be removed by treating deparaffinized and hydrated microscopic sections with one of these solutions:

  • absolute alcohol saturated with picric acid for 10 min-3 hours. then wash sections well with water

  • 100 mL 70% alcohol containing 3 mL ammonium hydroxide for 30 min-3 hours. then wash, rinse in 1% acetic acid and wash again

• mercury pigment removed as follows:

  • 1. treat deparaffinized and hydrated microscopic sections with Gram or Lugol iodine for 10 min

  • 2. wash sections in running water

  • 3. place sections in 5% solution of sodium thiosulfate for 3 min

  • 4. wash sections for 10 mins. and then stain

• Lugol Iodine solution

  • 1g iodine, 2g potassium iodide, 100 mL distilled water. Mix until dissolved