FIXATION PT1

PURPOSE OF FIXATION?

  • Alters tissue by stabilizing proteins

  • To preserve the morphology of the cells 

  • To prevent autolysis (self-destruction)

WHICH FIXATIVE IS BEST?

  • All fixatives have pros and cons; there will always be a trade-off

  • Most often, 10% neutral buffered formalin (NBF) is the fixative choice 



SOME QUESTIONS TO CONSIDER WHEN CHOOSING A FIXATION METHOD:

  • What molecular changes will the tissue undergo?

  • What components may be lost?

  • How well are cellular structures preserved?

  • Will it leave the tissue prone to shrinkage or swelling?

  • What effect will the fixative have on required staining methods?

  • What effect will the fixative have on required staining methods?

  • Cost and safety of fixative reagents?



METHODS OF FIXATION

  1. PHYSICAL FIXATION

  1. HEAT

  • Denatures and stabilizes proteins 

  • Most useful in speeding up other forms of fixation rather than a stand-alone method

  • Maximum temperature is 680C – if higher, nuclei will be overstained and pyknotic (small, shrunken)

  1. FREEZING

  • Tissue is freeze-dried by submerging in liquid nitrogen

  • Mostly used in research, rarely for clinical purposes

  1. DESSICATION

  • Air-drying of touch preps (a small amount of tissue is smeared onto a glass slide)

  • Most often preparations for Wrights stain

  • Rarely used for other applications



  1. CHEMICAL FIXATION 

  1. ADDITIVE 

  • Protein combines with fixative to make it insoluble (chemical link)

  • Common additive fixatives: mercuric chloride, chromium trioxide, picric acid, formaldehyde, glutaraldehyde, glyoxal, osmium tetroxide, zinc chloride or sulfate

  • May change the shape of the protein’s structure, and also the charge on the tissue

  1. NONADDITIVE

  • Mostly organic compounds (acetone and alcohols) that act on the tissue without combining with it 

  • Usually dissociate bound water molecules from tissue protein groups

  • Overexposure to non-additive fixatives can cause shrinkage and hardening of tissue

  1. COAGULANT

  • Create a network (mesh) within tissues – allows solutions to easily penetrate inside of tissue

  • Examples: alcohol, acetone, zinc salts, mercuric chloride, cupric sulfate, and picric acid 

  1. NONCOAGULANT

  • Creates a gel within tissues – so penetration by subsequent solutions is difficult

  • Examples: glutaraldehyde, glyoxal, potassium dichromate, osmium tetroxide, and formaldehyde (Go Get Plenty Of Fish)



QUALITY OF FIXATION:

  1. TEMPERATURE: increased temp increases the rate of fixation but also increases rate of autolysis in unfixed portions of tissue

  2. SIZE OF TISSUE: if tissue is too large, not enough may be exposed so it will not properly fix; must open or ‘bread-loaf’ large specimens to expose enough surface area; routine thickness for grossing ≤3-4mm to fit into cassette for processing

  3. VOLUME RATIO: 15-20x fixative volume to tissue size; additive fixatives deplete as they bind to tissue

  4. TIME

  • Exposure time, or cold ischemic time: time from interruption of blood supply until tissue goes into fixative (duration from extraction to fixative)

  • Duration, or how long the tissue remains in fixative before processing: minimum of 6-8 hours (for small specimens) formalin fixation before processing because if not fixed well, it will not process well and will not stain well; with many fixatives, the tissue cannot remain indefinitely without overhardening tissue

  1. PENETRATION RATE: (how fast the fixative will go through) coagulant fixatives have a maximum penetration once proteins are coagulated; formalin penetrates fast but continues to cross-link proteins for a long time after penetration; penetration is also affected by heat but not concentration 

  • Medawar constant: d = kt

d = depth of penetration, in mm

k = coefficient of diffusability (specific to the fixative)

t = time, in hours



FAST TO SLOW PENETRATION RATES OF SOME SIMPLE FIXATIVES:

  1. Formaldehyde (slower cross-linking)

  2. Acetic acid

  3. Mercuric chloride

  4. Methanol (and probably ethanol)

  5. Osmium tetroxide

  6. Picric acid



  1. STORAGE: may need to go back to “wet” tissue in storage for additional testing; can be stored indefinitely in formalin; for other fixatives, the tissue may have to be transferred to another solution for long-term storage

  2. pH: can affect fixation to an extent for light microscopy (LM); very important in electron microscopy (EM)

  3. OSMOLALITY: the number of particles in solution; not of high importance in LM, but very important in EM; should be in an isotonic state



REACTIONS WITHIN CELLS WITH FIXATIVES

  1. NUCLEUS: DNA/RNA and attached protein found in nucleus 

  • Acetic acid and Carnoy are preferred for nucleic acids as they fix cell nuclei 

  • Others likely trap DNA/RNA within stabilized nuclear proteins

  • Coagulant fixation results in sharper nuclear staining 

  1. PROTEINS: majority of non-nuclear staining is due to proteins; tertiary protein structure may be shifted by their reaction with fixatives, and charges on the proteins may be shifted by additive fixatives; this can influence their uptake of dye solutions 

  2. LIPIDS: only 2 will fix lipids so they are not lost in subsequent processing (osmium tetroxide is the best and chromium/chromic acid to a lesser degree); other fixatives may only preserve lipids 

  3. CARBOHYDRATES: some carbs are lost (water soluble) but with many fixatives, glycogen is retained as result of being “trapped” by the fixed proteins; a non-aqueous fixative such as alcohol is ideal for fixing glycogen



HALLMARKS OF PROPER FIXATION

On an H&E-stained slide, well-fixed tissue will demonstrate the following:

  • Well-defined nuclei, with nuclear membranes and chromatic patterns staining a crisp blue with hematoxylin

  • No smudginess, fading, or bubbling of nuclear material

  • No shrinkage or artifact spaces between cells

  • Cytoplasm of cells should stain well with eosin



FIXATION PROBLEMS

  1. DELAYED FIXATION: autolysis may occur–enzymatic tissue breakdown; result may be seen as absence of epithelial layer of tissue, or empty spaces around cells (artifact from cell shrinkage)

PREVENTION: ensure tissues are put into sufficient fixative ASAP after removal from patient; during grossing, opening and ‘bread-loafing’ while organs or resections to expose all parts/layers of tissue to the fixative used 

  1. INCOMPLETE FIXATION BEFORE PROCESSING:  separation of tissue components on water bath during microtomy; H&E-stained sections may show: poor tissue morphology, smudgy nuclear staining, no defined chromatin, nuclear bubbling artifact, ‘zonal fixation’ - different staining intensities of tissue periphery (formalin-fixed) than centre of tissue (fixation completed by processing alcohols)

PREVENTION: ensure sufficient fixation time has been given for type and size of tissue, and fixative type; consider a more rapid-acting fixative; ensure proper fixative:tissue ratio (20:1); ensure tissues are grossed no more than 3-4mm thickness, and that cassettes are not tightly packed



SIMPLE AQUEOUS FIXATIVES

  1. ACETIC ACID

  • Non-additive, coagulant

  • Fixes nuclei (precipitates/coagulates nuclear proteins and DNA) but non-coagulant for cytoplasmic proteins 

  • Penetrates rapidly

  • Leaves tissues soft

  • Swells proteins (tissue) because of acid pH-links in proteins are broken and water loving groups are exposed

  • Lyses RBCs

    • USES: in compound fixatives

    • SAFETY: add ACID TO WATER when preparing from concentrated solution and store at room temp, away from oxidizers, nitric acid, and strong caustics

  1. FORMALDEHYDE

  • Additive, non-coagulant

  • Reacts with group containing H+ to form methylene bridges that link protein chains

  • Pure formaldehyde is a colorless gas, when dissolved in water becomes a 37%-40% stock solution

  • In concentrated solutions, paraformaldehyde may be precipitated out from formaldehyde as a white powder; the addition of methanol counteracts this

  • Reaction with amino groups affects ability of certain structures to bind eosin; thus, tissue reactions are more “basophilic”

  • Lipids preserved but can be dissolved in processing, can do frozen sections of formalin fixed tissue if need to demonstrate

  • Glycogen is not fixed, but trapped in proteins

  • Penetrates quickly, but fixes slowly (cross-link form over time)

  • Less shrinkage than some other fixatives

  • Hardens more than all except ethanol

  • Relatively inexpensive, stable, and can do more special stains than with other fixatives

    • MOST COMMON FIXATIVE: 10% neutral buffered formalin (NBF); to get 10% formalin, the stock is diluted 1 in 10, which then contains 3.7%-4% formaldehyde

    • SAFETY: formaldehyde is a carcinogen; use in fume hood with proper PPE

    • DISPOSAL: reagent recycling, neutralization, or chemical disposal company

  • Formalin pigment: or black acid hematin; a dark brown crystalline pigment found if pH drops below 6.0 (creating formic acid), especially in tissues containing large amounts of blood; pigment can mask or simulate organisms; can interfere with silver stains, and is birefringent

    • PREVENTION: neutral pH (buffers the solution to control pH)

    • REMOVAL: treating tissue with alcoholic picric acid (then wash to remove yellow) or use alkaline alcohol

    • MOST COMMONLY USED: 10% NBF that is buffered with sodium phosphate “monobasic” and “dibasic”



DIFFERENT FORMULATIONS FOR VARIOUS APPLICATIONS:

  1. Zinc formalin

  2. Calcium formalin

  3. Saline formalin

  4. Alcoholic formalin



  1. GLUTARALDEHYDE

  • Additive, non-coagulant

  • Dialdehyde; one group on each of the molecule

  • Overhardens tissue

  • Incompatible with oxidizers and alkalis

  • Unstable and breaks down in oxygen so its prepared just before use

    • USES: for electron microscopy as it preserves ultrastructures well (buffer added)

    • SAFETY: sensitizer (respiratory, digestive, and skin irritation); use under fume hood

  1. GLYOXAL 

  • Additive, non-coagulant

  • Smallest dialdehyde

  • Usually 40% aqueous solution

  • Very rapid fixation

  • Alcohol or aqueous based

  • Reacts with oxygen containing end groups at one range of pH and with amine groups at another, slower to form crosslinks

  • Smaller molecule, aldehyde groups do not interfere with PAS stain like glutaraldehyde

  • Cannot use for Helicobacter pylori stains

  • Erythrocytes are lysed and granules in eosinophils dissolved

  • Not carcinogenic, but is an irritant

  1. MERCURIC CHLORIDE

  • Additive, coagulant

  • Very toxic, affects central nervous system

  • Absorbed through skin so rarely used

  • Corrosive, use is limited to compound fixatives

  • Inhibits freezing

  • Penetrates poorly with shrinkage/hardening of tissues

  • Leaves tissue receptive to staining

  • Mercury pigment: brown, bifringent; no prevention but removed by treatment by iodine, then sodium thiosulfate; iodine oxidizes mercury to mercuric iodide and then sodium thiosulfate removes excess iodide

  1. PICRIC ACID

  • Additive, coagulant

  • Does not fix lipid or carbs but may be used in compound fixatives for glycogen

  • Will decalcify small amounts of calcium 

  • Afterwards, tissues will be very sensitive to acid dyes so they should be washed out of the tissue before processing (use 50% alcohol with lithium carbonate)

  • Leaves tissue soft but allows significant shrinkage during processing (only one greater is ethanol)

    • USES: fixative and stain

    • SAFETY: very explosive when dry; moisture should be >10%

  1. OSMIUM TETROXIDE

  • Additive, non-coagulant

  • Mostly for EM

  • Fixes small amounts of fats in sections (thin sections, very poor penetration–only few cell layers deep)

  • Expensive, hazardous, and vaporizes readily

  • Does not work well with anionic (acid) dyes but good with cationic (basic) dyes

    • USES: to fix lipids in cell membranes so they become electron dense

    • SAFETY: in fume hood, will fix nasal mucosa, conjunctiva of eye if contact occurs

  1. POTASSIUM DICHROMATE

  • Additive, non-coagulant

  • Rarely used alone

  • Attaches to some lipids but does not preserve 

  • Softens and shrinks tissue after processing

  • Reacts with both COOH- and OH- groups on proteins; increases number of basic (+) groups available for dyes; tissue more affinity for eosin (anions)

    • SAFETY: very toxic (chromium); carcinogen and corrosive; use in fume hood

  • Chrome pigment: treat with running water before processing to prevent pigment or if it has formed, remove/reduce pigment with acid alcohol treatment

  1. ZINC SALTS

  • Additive, coagulant 

  • Zinc sulfate (moderate health risk-respiratory irritant) is an acceptable substitute for mercury 

  • May be better than NBF for IHC staining (no retrieval required)

  • Zinc chloride (severe health risk-corrosive) may not be allowed on processors (corrosive)

  • Precipitates can form when zinc comes into contact with alcohols on processor; can be removed with dilute acetic acid; prevention involves washing tissues well in water before dehydration



NON-AQUEOUS SIMPLE FIXATIVES

  1. ALCOHOLS

  • Non-additive, coagulant

  • Absolute ethyl or methyl alcohol used

  • Dissolves fat

  • Overhardens, shrinks, and dehydrates

    • USES: for touch preps/blood smears, for preserving water-soluble targets in tissues (such as glycogen and urates)

  • SAFETY: highly flammable; store in fireproof cabinets

  1. ACETONE

  • Non-additive, coagulant 

  • Causes significant shrinkage, hardening, and distortion of tissue–not a good general fixative

  • Rapid fixation and carried out cold (40C)

    • USES: for enzyme demonstration, fixing brain tissue for rabies investigation, and frozen sections for immunohistochemical techniques 

    • SAFETY: highly flammable, store in fireproof cabinets, low flash point (40C)








ADDITIVE

NON-ADDITIVE

COAGULANT

Mercuric chloride

Chromic acid

Picric acid

Zinc salts

Cupric salts

Alcohols

Acetone

Acetic acid (nucleic acids only)

NON-COAG.

Glyoxal

Glutaraldehyde

Potassium dichromate

Osmium tetroxide

Formaldehyde




SIMPLE FIXATIVES THAT CAN FORM A FIXATION PIGMENT IN TISSUES:

  1. Formalin

  2. Mercuric chloride

  3. Potassium dichromate