Topic 6 - Tissue Processing and Embedding

What is tissue processing

Any steps taken to prepare tissue for microtomy.

4 stages of tissue processing

Fixation, Dehydration, Clearing, Infiltration

What is dehydration

Stage where dehydrating agents remove all unbound water and fixative from tissue. Unbound water is the intracellular fluid and interstitial fluid

Major functions of clearing

Removes dehydrating agent and acts as solvent for the paraffin wax used for infiltration.

Also raises the refractive index of the tissue closer to that of glass, allowing for high power magnifiication

What is infiltration

Tissue impregnated with a support medium, usually paraffin wax, allowing for cutting of thin sections. After this stage, the tissue is ready for embedding

Dehydrating solution characteristics

Hydrophillic solutions that draw water out of tissues. The solutions are exchanged for increasingly concentrated solutions to dilute reminaing water

Most commonly used dehydrating agents

Ethanol, Isopropyl alcohol, Methanol

Dehydrating properties of methanol

Routinely used.  Infinitely soluble with water, fast-acting, and relatively non-toxic. May cause hardening and shrinkage of tissue specimens.

Dehydrating properties of isopropyl alcohol

Miscible in water, but immiscible in salt solutions, so tissue must be washed following fixation. Isopropyl alcohol can be mixed with molten paraffin, so clearing agents are not always used/required. Relatively non-toxic, and does not harden or shrink tissue.

Methanol dehydrating properties

Similar to ethanol, but more hazardous (toxic).

Process of dehydration

Tissue subject to 50-70% alcohol immediately after fixation, followed by increasingly concentrated solutions (80, 90, absolute alcohol)

Waht makes a good clearing agent

Soluble in both dehydrants and paraffin

Role of clearing agent

Once tissue is dehydrated, clearing agent dissolves remaining alcohol, leaving tissue receptive to molten wax (non-polar)

Clearing agent refractive index

High refractive index (1.4-1.51), allowing the tissue to appear transparent when mounted on glass

Common clearing agents used in the histo lab

Xylene, Toluene, Chloroform, Xylene Substitute

Clearing agent properties of xylene

Routinely used. Fast-acting, and miscible with most solvents as well as paraffin. Over-exposure will harden tissue. Flammable and moderately toxic.

Clearing agent properties of toluene

Similar in action to xylene, but unlikely to harden tissues. More volatile than xylene (resulting in more vapours).

Clearing agent properties of chloroform

Sometimes used for C.N.S specimens that may become brittle when cleared with xylene (especially brain and eyes). Non-flammable, but highly toxic. Chlooform in the presence of oxygen may form phosgene, which is exceptionally hazardous (a dangerously poisonous gas).

Xylene substitute

Short-chain aliphatic hydrocarbons (the same class of chemicals as butane, petroleum jelly, and paraffin wax). They are less toxic and work almost as well as xylene. They are intolerant of water, so the final alcohol must be completely anhydrous.

What are universal solvents + examples

• Solvents that are capable of dehydrating + clearing tissue

  • Most common are Tetrahydrofuran (THF) and dioxane

Role of infiltrating media

Forms a matrix and prevents distortion of tissue durig microtomy. Most common is paraffin wax

Common additives to paraffin wax

Plasticizers, Beeswax, Rubber, Resins

Role of plasticizers in paraffin wax

Used to make wax harder + facilitate ribboning

Role of beeswax in paraffin

Lowers melting point and makes sections sticky

Role of rubber in paraffin wax

Facilitates ribboning by increasing elasticity

Role of resins in paraffin wax

Makes wax harder/increasing melting point

Factors that effect tissue processing

Agitation, Heat, Vacuum/Pressure, Viscosity

How does agitation affect tissue processing

Ensures solutions surrounding tissues are not locally saturated, and helps avoid dead zones where solutions are not exchanged during processing.

How does heat affect processing

Speeds up processing, but must be careful as excess heat distorts microscopic details of delicate tissues. Recommended heat is the minimum amount to keep wax molten

How does vacuum/pressure affect processing

 Pressure can force solutions into tissues, and vacuum can be used to “open” porous structures. The vacuum is particularly effective as an aid during infiltration; by lowering the pressure in the retort, the clearing agent is more easily replaced by hot wax, and the wax can more easily penetrate tissue structures.

Affect of viscosity on processing

Lower viscosity solvents work more quickly than wax

Pros + Cons of microwave processors

Pros: Improves turn around time, formallin + xylene free

Cons: Cost

Drawbacks of xylene-free substitutes

Do not clear alcohol as quickly as xylene, and is less tolerant of water contamination (ethanol must be 97% pure to be effectively cleared)

5 steps of processing with no clearing agent

• Fixation

• Ethanol dehydration (50% ethanol/water blend)

• Continued dehydration/clearing (80% ethanol/20% isopropanol)

• Isopropanol clearing (100% isopropanol)

  • High-temperature paraffin wax infiltration – the first wax bath should be held at 85 °C to effectively eliminate isopropanol (B.P = 82.5 °C at 760 mmHg)

How are repeating reagents used to make sure they are the least contaminated

They are rotated to ensure the last station is the least contamination, and the first station in the series is discarded

How to remove paraffin from instrument lines

Flush lines with xylene to dissolve the wax and the flush lines with alcohol to dissolve the xylene

Most common cause of inadequate processing

Incomplete dehydration. Microtomy blocks have soft, mushy areas at the center of the tissue (where wax did not infiltrate). This creates a hole on the finished slide

What is the effect of a tissue that is over-processed

Tissue is brittle, and fragments easily. Microchatter is seen microscopically

What is embedding

Once tissue has been fully impregnated by paraffin, it is oriented in a mold where more wax is added and the block is left to cool in order to preserve the desired orientation. This provides a support medium for microtomy, facilitating the cutting of thin sections. It should also provide elasticity to resist the compression caused by microtomy.

Role of hard embedding wax

Higher melting point, and supports harder tissues. Can cut thinner sections

Role of soft embedding wax

Easier to ribbon, making them ideal for serial sectioning

Procedure of embedding

1. The tissue is removed from the cassette and examined to determine if any particular orientation is required.

2. A suitably sized mold is chosen (around 2-3mm bigger than the tissue on all sides to ensure good support), which is then partially filled with wax.

3. The specimen is placed centrally in the mold and transferred to a cold plate

4. . The tissue is gently held down in its desired orientation until a thin layer of wax at the bottom of the mold hardens, becoming opaque.

5. The cassette (with patient identifier) is placed on top of the mold, and additional wax is added. It is important that the wax fills all spaces in the tissue (otherwise the tissue will collapse during sectioning in those areas).

6. The block is now finished, and is placed on the cold plate until it is completely soli

Most critical step in embedding process

Orientation

Effects of improperly oriented tissue in embedding

Misdiagnosis (pathologist does not see area of interest, or the relationship between one tissue layer and another)

How to ensure specimen is embedded in correct orientation

Grossing person records instructions, or uses ink to indicate which side is placed downward

How should tissues with lumens be embedded

 embedded to show a cross-section of the lumen.

How should tissues with layers be embedded

should be embedded “on-edge”, so that all layers are demonstrated on each slide

How to embed multiple pieces of tissue in the same cassette

placed side-by-side, and oriented with the epithelium facing the same direction. When embedding curettings, cluster them centrally, but do not overlap

How to improve microtomy when embedding an elongated tissue

 offset the tissue slightly (up to a 30° angle from horizontal). This is especially important for tougher/harder tissues (bone, uterus) to prevent compression-type artifacts.

Paraffin block storage

Once a block has been cut and the slides are satisfactory, the block is sent for storage. Unlike the “wet” specimens (those stored in formalin that are discarded after 8 weeks), paraffin blocks are stored indefinitely.

How long should paraffin blocks be retained prior to discard

at least 30 years prior to discard. Blocks are routinely stored longer than that; it is dependent on the amount of storage space available.

Use of acrylic resins in histology

Section undecalcified bone, teeth, or to cut thin sections (less than 2µm) for light microscopy.

Examples of acrylic resins

Methyl methacrylate (MMA) or glycol methacrylate (GMA)

Can you use acrylic resins to embed for electron microscopy

No, they are unstable in eelctron beam

What resins should you use for embedding for electron microscopy

Epoxy resins. Also ideal for cutting semi-thin and ultra thin sections

Dangers of working with resins for embedding

use of potentially dangerous chemicals including organic peroxides (poisonous and potentially explosive). The setting/hardening of these resins involves a strongly exothermic reaction which can become hazardously hot. Always work in a fume hood to control the release of toxic vapours (carbon monoxide, nitrogen oxides, and ammonia are most notable).