Tissue Processing and Fixation

Processing

steps needed to dehydrate, clear, and infiltrate tissue so that it may be sectioned and placed onto slides

method is chosen based on the tissue types, requiremnt of special techniques, or the preference of the pathologist

Processing steps

fixation, dehydration, clearing, and infiltration

Dehydration

uses hydrophillic chemical solutions in order to draw water out of the tissue

can use alcohol, acetone, or universal solvents

Overdehydration

results in hard, brittle tissue

can be difficult to sectioning

Inadequate dehydration

prevents effective clearing and poor infiltration with the paraffin

results in soft, mushy, blocks and is a major source of processing related problems

Ethanol - dehydration alcohol

the most common and inexpensive reagent that mixes well with water

goes through graded concentrations (70%, 95%, and 100%)

Methanol - dehydration alcohol

metabolizes as formeldahyde and used to create reagent grade alcohol from ethanol

used to fix blood slides/smears

Isopropanol - dehydration alcohol

doesn’t harden or shrink tissue like ethanol

doesn’t dissolve common stains like eosin, meaning ethanol will be needed eventually

Acetone - dehydration

effective and fast acting

has more disadvantages than advantages, causes shrinkage

Universal solvents - dehydration

can be used for dehydration or clearing

includes dioxin, tertiary butanol, and tetrahydrofuran

disadvantages (tissue distortion) make it not common

Clearing

removes dehydration agent, also called dealcoholization agent

makes tissue receptive to infiltration agent

must be miscible with the dehydration and infiltrating agent

Xylene - clearing agent

most widely used bc its effective in replacing ethanol and msicible with paraffin, but doesn’t mix with water

overhardens tissue if left for too long, requires proper disposals

Toluene - clearing agent

aromatic hydrocarbon thats more water tolerant

doesn’t overharden tissue

Benzene - clearing agent

rapid acting hydrocarbon

volatile and requires constant topping off in machine

Chloroform and acetone - clearing agent

chloroform is slow acting with disadvantages including moisture absorption and formation of poisoness gases when heated

acetone is used as a universal solvent, but is volatile

Essential oils - clearing agent

derived from plants but expensive to produce

has a harsh odor and is difficult to work with, used in special studies

Limonene and aliphatic hydrocarbons - clearing agent

limonene is used as a xylene replacement but has a harsh smell and causes allergies while reducing the hardening and shrinking of the tissue

aliphatic hydrocarbons are lower in reactivity but don’t tolerate high humidity or moisture

Infiltration

tissue must be dehydrated abd cleared prior

paraffin is traditional, but other means can also be usedPar

Paraffin wax - infiltration

holds cells and intracellular structures intact during sectioning

versetile in degree of stickiness, adhesion, brittleness, hardness, and level of support depending on needs

melting point between 55 and 58 C

Carbowax - infiltration

water soluble and can infiltrate tissue directly without dehydration or clearing

doesn’t disolve fats and they can be seen in sections

sections dissolve in water baths, blocks must be chilled prior to sectioning due to solubility

Glycol methacrylate - plastic infiltration

acrylic resin that is miscible with water

provides support for hard tissue and allows for thin sections with well pressured cellular details (bone marrow, kidneys, lymph nodes)

staining is difficult due to tissue remaining in the media

Epoxy resin - plastic infiltration

used for electron microscopy and requires thin sections

uses transitional fluids and undergoes polymerization

requires a dimond knife

Celloden - infiltration

any type of nitrocellulose

used in research and neuropathy

staining and sectioning is tedious

blocks are hardened with chloroform and can be cut wet or dry

Agar and gelatin - infiltration

produces a single block of friable tissue or multiple fragments

useful for frozen sections from the cryostat

allows for multiple pieces to be embedded in a single block

Factors affecting tissue processing

tissue size - biopsy versus resection time

tissue density - lung versus breast

lipid content - longer penetration time

tissue thickness

Embedding

tissue is placed into the mold and then filled with paraffin

placed at a slight angle to ensure blade makes contact with smaller areas

orientation is important

Things to consider when embedding

the plane of the tissue and how it would be cut/viewed

type and size of tissue as well as the number of pieces

how the pathologist will view the tissue after embedding

types of tissue - embedding

tissue types have different orientations due to their composition

multiple layers need to be shown top to bottom

tubular structures need to be embeeded to show outer surface and lumen in a single section

size of tissue - embedding

large flat sections are embedded as they are found in the cassette

small pieces can be shuffled during processing

number of pieces - embedding

single sections should be placed in the center

medium pieces should be organized to the long axis in the order they were placed into the cassette