Chapter 4 - Fixation

Fixation

What happens to tissue as soon as it is removed from the body?

Death of the cell/tissue is recognized with the lack of blood flow and/or nerve impulses.

What does autolysis means?

Literally it means “self-splitting”. Enzymes in the cell start to digest the cell, destroying structures of nuclei, cytoplasm, organelles, and membranes.

What is putrefaction?

Is the further tissue breakdown by bacteria

What prevents autolysis and putrefaction from occurring?

Fixation

What does fixation do for the tissue?

It allows the cellular morphology to be saved. Also important, the fixative transforms soluble cellular components into insoluble components so that they are not lost in the subsequent tissue processing protocol.

How can fixation work?

It can perform what it does by the process of denaturation.

What is denaturation?

Denaturation is the change in the 3D structure of the macromolecules. Generally applied to proteins (enzymes are also proteins) or nucleic acids. When enzymes have their 3D structure change, the enzymatic activities are lost. The change in conformation of a protein also changes its solubility

What’s the older definition of fixative action?

A fixative kills, penetrates, and hardens tissue.

Why is penetration important in fixatives?

Because adequate penetration of the fixative ensures fixation of the interior of the tissue, as well as the few exterior cell layers.

What does hardening or firming of tissues during fixation provide?

It provides the tissue the rigidity necessary to withstand the subsequent tissue processing.

How does protein denature postmortem?

Putrefaction (bacterial attack) and autolysis (enzyme attack)

How to prevent bacterial attack apart from fixation?

By observing very strict antiseptic techniques

Can autolysis be prevented without fixation?

No

Why does autolysis occur?

Because some of the enzymes present in tissue continue their metabolic processes, even after interruption of the blood supply, until something happens to stop the enzyme action.

Can severely autolyze tissue still stain?

No

Fixation helps maintain proper relationship between cells and what extracellular substances?

Connective tissue fibers like collagen, reticulin, and elastin.

A well tissue is almost impervious to ____ during the ______ and ______ prcedures.

• abuse

• processing

• staining

A fixative also functions to bring out the differences in ___________ and to increase the visibility of, or the contrast between, different tissue ______

• refractive indexes

• elements

Does fixation enhance or hinders staining?

Enhance

What is the exceptions that will make staining poor by fixation?

The masking of antigenic sites by fixation can lead to faint or negative immunohistochemical staining. But antigen retrieval techniques can offbalance this.

Does fixation make cell constituents soluble or insoluble?

Insoluble. With tissue proteins serving as the primary target for stabilization

Most of the time, fixation will make these components of the cell lost, but some fixatives can help stabilize or retain these. What are these?

Lipids and carbohydrates,

What is heat fixation?

When you use a microwave to fix tissue by breaking internal bonds, making the molecule unwind, and new bonds create to stabilize the molecule.

What is Desiccation?

Another physical method of fixing protein, but rarely used if ever in histopathology.

What is the most common used for desiccation fixation?

Air drying of touch preparations for Wright staining

How are reagents classified?

• Additive or nonadditive

• Coagulant or noncoagulant

How does additive fixatives work?

Chemically link, or add themselves, to the tissue and change it.

What happens when a fixative molecule adds onto a tissue macromolecule?

The electrical charge at the site of attachment may be changed

What are the common additive reagents?

• mercuric chloride

• chromium trioxide, picric acid

• formaldehyde

• glutaraldehyde

• glyoxal

• osmium tetroxide

• zinc sulfate or chloride

What are nonadditive fixatives and how do they work?

Predominantly organic compounds like acetone and the alcohols. They act on tissue without chemically combining with it. The primary mechanism of action is to dissociate bound water molecules from tissue protein groups.

What happens when bound water is removed from tissues?

The solubility of the protein is lost and it precipitates. Excessive removal of bound water results in shrinkage and the hardening.

What are coagulant fixatives and their effects on tissue?

Precipitate or coagulate proteins by disrupting their hydrophobic interactions or hydrogen bonds. They create a porous, mesh-like structure that allows for good penetration of stains and reagents.

What are the advantages and disadvantages of coagulant fixatives

Advantages:

• rapid penetration

• Good for preserving cytoplasmic components and fine cellular detail

Disadvantages:

• Can cause tissue shrinkage or hardening

• Poor preservation of ultrastructure

What are noncoagulant fixatives and their effect on tissue?

They form covalent bonds between proteins and other molecules, stabilizing the tissue. They make tissue retain structural integrity of cells and tissues more completely.

What are the advantages and disadvantages of noncoagulant fixatives:

Advantages:

• Excellent preservation of tissue architecture

• Minimal shrinkage

Disadvantages:

• Slower penetration

• Cross-linking may mask antigen sites (problems for IHC)

What are some common coagulant fixatives?

• zinc salts

• mercuric chloride

• cupric sulfate

• ethyl alcohol

• methyl alcohol

• acetone

• picric acid

What are common noncoagulant fixative reagents?

• formaldehyde

• glutaraldehyde

• glyoxal

• osmium tetroxide

• potassium dichromate

• acetic acid

What are all the factors that affect fixation?

• temperature

• size

• volume ratio

• time

• choice of fixative

• penetration

• tissue storage

• pH

• osmolality

In what way does temperature affect the tissue in fixation?

Affects tissue morphology.

In general, an increase in temperature increases the rate of fixation BUT also increases the rate of autolysis ad diffusion of cellular elements.

What is the traditional temperature for fixation?

0C - 4C is ideal for specimens for electron microscopy.

Today though, higher temps are used and in general increasing the temp of fixative to ~45C is reported to have little effect on morphology

How should size of specimen be considered when fixing tissue?

If large specimens such as segments of colon or SI are held for an extended period without being surgically opened to expose all layers, the fixative will have difficulty penetrating through the entire wall to the inner epithelial surface.

What’s the result of not surgically opening large tissues in fixation?

Frequently results in autolysis of the epithelium

How should one do with solid organs like spleen or kidney?

They need to be “bread-loafed” as soon as possible and covered with adequate fixative.

What should the fixative volume be compared to the tissue?

At least 15-20X greater than the tissue.

What does the diffusion of the fixative into the tissue displace?

The free water and serum proteins. This 2-way exchange does not affect fixative as long as a large volume is used. If not, then it compromises the fixative composition.

How is time important in fixation?

Ischemic time and duration.

What is ischemic time?

The interval between interruption of the blood supply to the tissue in fixative. Ideally, the tissue should be placed immediately after it is removed or immediately after death.

What’s the difference between these two samples?

The one on top is mucosa that’s been excellently preserved of SI. No autolysis present and epithelium intact.

The one at the bottom was delayed for fixation. Marked autolysis seen except for a few glands, or the cryps, the epithelium is gone. Most goblet cells and argentaffin cells have disappeared. Only the denuded lamina propria of the villi can be seen.

What’s the difference between these two samples?

The one at the top has not been completely fixed before processing. Shows poor stabilization of tissue structures, and proper relationships are not maintained.

The one at the bottom was well fixed before processing, shows proper relationships of tissue structures. The interalveolar septa are well preserved and the alveolar sacs are clearly seen.

How is the duration of the fixation important?

Adequate fixation time is of primary importance in quality assurance. Tissue that has not been fixed well does not process well, and will not stain well.

How long should formalin have to act before the remainer of the processing steps take place?

6-8 hours at least

What to do when you don’t want tissue fixation like for immunofluorescence, or enzyme profile?

Tissue should be frozen

What fixative allows most tissues elements to be demonstrated with special techniques?

Formalin fixation

What fixative to use in the specific scenarios when you need muscle cross-striations are to be stained with phosphotungstic acid-hematoxylin?

Zenker or Bouin solution

What fixative to use when the tissues are to be stained with trichrome technique?

Buoin solution

What is postfixation or mordanting?

To increase the staining reaction, a tissue section of tissue that has been fixed with 1 reagent frequently can be treated with another fixative reagent. Used in Masson Trichrome technique, in which a tissue section of formalin fixed tissue is mordanted with Buoin solution before staining.

Some tissue elements cannot be demonstrated if the original fixation is incorrect, like chromaffin granules. Explain

Chromaffin granules, found in cells of adrenal glands, are helpful in the identification of pheochromocytomas, but these granules cannot be demonstrated after formalin fixation.

How to fix for the demonstration of chromaffin granules?

Primarily dichromate fixative such as Orth solution. Urate crustal are water soluble and require a nonaqueous fixative such as absolute alcohol.

What is penetration if fixation?

A physical process and fixative solutions penetrate at vastly different rates.

What is the formula that establishes how fixatives penetrate tissues?

d=k sqr root(t)​

d = depth of penetration

k = coefficient of diffusibility; called the Medawar constant or K value. Fixative-specific constant.
t = time

What is the K value for 10% NBF?

K = 0.78. This means that 10% NBF would penetrate 1 mm per hour. So for example, to get to the center of a 4 mm grossed experiment (2 mm mark) the time would 2² = 4 hours.

Factors that influence penetration rate?

The K value increases as the rate of penetration decreases

Distance increases with the time.

Increase in pressure also improves formalin penetration

Type of tissue

According to Baker, what are the factors that determine the minimum length of time of fixation?

Rate of penetration and mode of action.

When do most coagulant fixatives achieve their full effect?

At any particular depth as soon as they have penetrated to that depth at a concentration sufficient to cause coagulation.

List of fixative ingredients, in order of decreasing speed of penetration:

1. formaldehyde

2. acetic acid

3. mercuric chloride

4. methyl alcohol

5. osmium tetroxide

6. picric acid

Ethyl alcohol probably penetrates at a similar rate to methyl alcohol, but information is not available

T/F: The rate of penetration is not affected by heat

False. Rate of penetration is affected by heat.

Why is most of the fixation of larger specimens may be primarily the result of only 1 chemical in a compound fixative?

Because of the varying rates of penetration, and because fixation begins at the periphery of the tissue and proceeds inwards.

Why is the method of wet tissue storage important?

Because wet tissue often will be needed for additional studies, which may be impossible if tissue has not been fixed and stored properly.

Why is storage not usually a problem with tissue fixed in NBF?

Because the tissue may remain in this solution indefinitely, and this not true of many other fixatives. However, if IHC stains are anticipated at a future time, tissue should be transferred from formalin to 70% alcohol to stop crosslinking.

What is Osmolality?

Refers to the number of particles in solution and is not as important in light microscopic studies as in ultrastructural studies. A 1-Osm solution may be defined as 1 formula weight of a nondissociating compound (sucrose) per 1000 g of solution. Whereas one formula weight of a dissociating compound (sodium chrolide) per 1000 g of solution is equal to a 2-Osm, or 2000 mOsm solution.

What is the osmolality of body fluids?

~340 mOsm or 0.3 Osm

What does the terms isotonic, hypotonic, and hypertonic mean and why are they important in the context of fixation?

They refer to the osmolarity (solute concentration) of a solution relative to the inside of a cell. This is important because they will tell us what will happen to the cell and thus influence its morphology, especially in the preservation of cell volume and membrane integrity. Water will move outside or inside the cell to equilibrate the solute concentrations. Water moves from the place of lower solute concentration to higher solute concentration in order to even out cell and environment.

1. If a tissue is place in an isotonic solution, it means that the solute concentration is the same as the cell and its shape and volume are maintianed.

2. In a hypotonic solution, means that the solute concentration compared to the cell is lower, and that the cell has a higher solute concentration. This means water will go into the cell, the cell swells and may burst.

3. In a hypertonic solution, means that the solute concentration compared to the cell is higher than the cell, and that the cell has lower solute concentration. This means water will go out of the cell, the cell shrinks.

For an aqueous fixative, what is the most rapidly penetrating component? And what does this mean for the cell and how this is combated?

Water is the most rapidly penetrating component. This means that the central parts of a specimen are probably in contact with a hypotonic solution before fixation occurs. Unreactive salts with small rapidly diffusing ions (sodium sulfate or sodium chloride) frequently are added to fixative mixtures to prevent the damage caused by these hypotonic solutions.

Why does 10% neutral-buffered formaldehyde appear hypertonic (~1800 mOsm) but not affect cells osmotically?

Because formaldehyde is osmotically inactive, so despite the high measured osmolarity, it doesn't cause osmotic stress on cells.

What to do with biopsy tissue that cannot be fixed immediately?

Dampen a piece of gauze with saline solution, squeeze out the excess, and place the tissue on the dampened gauze. This treated in this way can be sealed in a plastic container and placed on ice for short-term holding.

What components are in the nucleus?

DNA (deoxyribonucleic acid), RNA (ribonucleic acid), and attached protein histones

What fixatives are preferred for nucleic acids, and which one does not react?

Acetic alcohol and Carnoy solution are preferred fixatives for nucleic acids; formaldehyde does not react with DNA and RNA in their native states until temperature reaches ~45C for RNA and 65C for DNA molecules by the fixed or stabilized nuclear proteins.

What is nuclear bubbling and how is it impactful?

Following formalin fixation, the nuclei often show coalescence of the chromatin into strands with intervening clear spaces. This can compromise the pathologic diagnosis.

When does nuclear bubbling potentially occur?

According to Banks, it is introduced in the DP step of formalin fixed tissue, because the nuclei are only delicately fixed.

Dapson attributes nuclear bubbling to the specimen being incompletely fixed in NBF before beginning dehydration.

Why does most nonnuclear staining occurs?

Because of the proteins present and the particular chemical group(s) with which a fixative reacts.

What are the 4 structures of proteins:

1. primary - amino acid sequence

2. secondary - conformation of the major polypeptide chain (backbone). Common shapes are alpha helix or beta pleated sheets.

3. tertiary - complete 3D shape of the protein and is mainly determined by the interactions of the side chain (R groups) of the amino acids

4. quaternary - applies to proteins that have multiple polypeptide chains that are joined together.

What happens when a fixative molecule adds onto a tissue macromolecule (additive fixative)?

The electrical charge is changed. If the electrical charge is changed, and that charge was a force helping to maintain the conformation of the protein, then the tertiary structure may be significantly altered. Thus, the function of degrading enzymes is stopped, preventing autolysis as well as inhibition of bacterial function (putrefaction).

Although several of the fixatives will preserve lipids, what fixatives will insolubilize lipids?

Osmium tetroxide and chromic acid

What is osmium tetroxide and what does it react with?

It is an additive and crosslinking fixative. It reacts with double bonds of unsaturated lipids. After cross-linking, it produces a dark brown color and is able to work as a stain as well as a fixative.

The retention of carbohydrates (eg, glycogen) as well as some lipids and nucleic acids are thought to result from:

entrapment in the crosslinked proteins.

What are the water based, or aqueous fixatives, and fixative ingredients that are discussed in the text:

1. acetic acid

2. formaldehyde (formalin alcohol included)

3. glutaraldehyde

4. glyoxal

5. mercuric chloride

6. osmium tetroxide

7. picric acid

8. potassium dicrhomate

9. zinc salts

10. others

Acetic acid formula

CH₃COOH

What’s the name for concentrated acetic acid and why

Glacial acetic acid because of its unusual freezing point of 16.6C.

What does acetic acid do?

It does not fix or destroy carbohydrates, and it does not fix lipids. It penetrates very rapidly and leaves tissues very soft. The major use of acetic acid in fixatives is the precipitation and preservation of nucleoproteins and DNA.

How does Baker classify acetic acid?

As a noncoagulant, stating that its precipitating action on nucleoproteins is different from that on nonnuclear proteins.

What is characteristic of acid fixation as long as the solution pH < 4.0?

Swelling. Collagen swells dramatically near a pH of 2.5 because links in proteins are broken and any hydrophilic groups present are exposed. Thus, water is absorbed.

What does acetic acid do to RBC?

TBCs are lysed by acetic acid

How should acetic acid be stored?

Room temperature and away from strong oxidizers, nitric acids, and strong caustics.

What can acetic acid cause?

Severe burns and has a permissible exposure limit of 10 ppm. Therefore, it should be transported in an acid carried or used under hood.

Should acetic acid be added to water or should water be added to acetic acid?

Acetic acid should be added to water, like with all dilutions.

How to prepare a 10% formalin solution

Dilute 1 part of the stock solution (37% formaldehyde) with 9 parts water. The resulting solution is 10% formalin or 3.7%-4.0% formaldehyde.

What is the most widely used fixative?

10% formalin

What are the safety issues with 10% formalin as a fixative?

In aqueous solution, formaldehyde combines chemically with water to form methylene hydrate (methyl glycol. HO-CH₂-OH). Methyl hydrate has a strong tendency to form short polymers of 8-10 units, and it has the same reactivity as formaldehyde. In 10% buffered solutions, the monomer predominates.

What do manufacturers do to prevent the formation of paraformaldehyde?

Add 10%-14% methanol to 37%-40% formaldehyde.

Why is paraformaldehyde used in many electron microscopy laboratories?

Because it yields a pure formaldehyde solution. The paraformaldehyde is purchased as a white powder and used as a 4% solutions, which is equivalent to a 10% formalin solution.