Tissue processing is a series of physical and chemical steps that prepare tissue samples for microscopic examination. It ensures the preservation of tissue structure and enables the production of high-quality diagnostic slides. Proper processing is critical for accurate diagnosis and research.

The key steps are:
Fixation – Stabilizes and hardens tissue.
Dehydration – Removes water and fixatives.
Clearing – Removes dehydrating agents and prepares tissue for infiltration.
Infiltrating – Permeates tissue with a support medium (e.g., paraffin wax).
Embedding – Orients tissue in a medium for sectioning.

Each tissue sample is assigned a unique accession number or code, which accompanies it throughout processing. Proper labeling ensures accurate identification and prevents mix-ups, which is critical for patient diagnosis and research.

The rate is influenced by:
Agitation – Increases fluid exchange.
Heat – Speeds up penetration but must be controlled to avoid tissue damage.
Viscosity – Lower viscosity speeds up fluid exchange.
Vacuum – Enhances infiltration and reduces processing time.

Agitation increases the flow of fresh solutions around the tissue, improving fluid exchange. It can reduce overall processing time by up to 30%.

Heat increases the rate of penetration and fluid exchange. However, excessive heat can cause tissue shrinkage, hardening, or embrittlement. Temperatures should not exceed 45°C to avoid damaging the tissue.

Lower viscosity (smaller molecules) allows faster fluid penetration, while higher viscosity (larger molecules) slows down the process. Most processing solutions have similar viscosities, except for cedar wood oil.

Vacuum enhances the infiltration of reagents into the tissue, reducing processing time. It also helps remove trapped air from porous tissues. However, excessive vacuum pressure can damage tissues.

The stages are:
Fixation – Stabilizes tissue.
Dehydration – Removes water.
Clearing – Prepares tissue for infiltration.
Infiltrating – Permeates tissue with a medium (e.g., paraffin wax).
Embedding – Orients tissue in a solid medium for sectioning.

Fixation stabilizes proteins and prevents tissue degradation by inactivating enzymes. It ensures minimal distortion of cells and prepares tissues for further processing. Incomplete fixation can alter staining results.

The most common fixative is 10% neutral buffered formalin (NBF). Other fixatives include Bouin’s solution (picric acid-based) and Carnoy’s fluid (alcoholic fixative).

For Bouin’s solution, tissues are placed directly into 70% alcohol to remove water-soluble picrates. For Carnoy’s fluid, tissues are placed directly into 100% alcohol.

Dehydration removes water and fixatives from tissues using graded alcohols (e.g., ethanol, methanol). It prepares tissues for clearing and infiltration by making them receptive to non-aqueous media.

Common dehydrating agents include:
Ethanol
Methanol
Isopropyl alcohol
Acetone
Butanol

Ethanol is a fast-acting, reliable dehydrant that is hydrophilic and miscible with water. It is commonly used in graded concentrations (70%, 95%, 100%) for dehydration.

Ethanol – Fast-acting, reliable, and commonly used.
Methanol – Highly toxic but can substitute ethanol.
Isopropyl alcohol – Used in microwave processing, causes less hardening and shrinkage.

Universal solvents (e.g., dioxane, tertiary butanol) both dehydrate and clear tissues. They are no longer used due to their hazardous properties and tendency to harden tissues.

Clearing removes dehydrating agents and prepares tissues for infiltration by making them translucent. It acts as an intermediary between dehydration and infiltration.

Criteria include:
Rapid penetration and removal of dehydrating agents.
Ease of removal by paraffin wax.
Minimal tissue damage.
Low flammability, toxicity, and cost.

Common clearing agents include:
Xylene
Toluene
Chloroform
Limonene reagents (citrus fruit oils)

Advantages: Fast-acting, recyclable, and commonly used.
Disadvantages: Can harden tissues with prolonged exposure and is flammable.

Xylene substitutes are aliphatic hydrocarbons (short- or long-chained). Short-chained substitutes evaporate like xylene, while long-chained ones are slower and may contaminate paraffin wax.

Limonene reagents are non-toxic, derived from citrus rinds. They are environmentally friendly but can cause sensitization and have a strong odor. They are not recyclable.

Paraffin wax infiltrates and embeds tissues, providing structural support for microtomy. It solidifies quickly, allowing thin sections to be cut without distortion.

Paraffin wax has a wide range of melting points (47–64°C), is inexpensive, and provides good support for sectioning. It is compatible with most stains and immunohistochemistry protocols.

Additives like plasticizers or resins modify the hardness of paraffin wax, making it suitable for different tissue types. However, excessive additives can make tissues brittle.

Alternatives include:
Resin – For electron microscopy and undecalcified bone.
Agar – For small, friable tissues.
Gelatin – For frozen sections.
Celloidin – Rarely used due to special handling requirements.

The tissue is oriented in a mold, covered with molten paraffin wax, and allowed to solidify. The wax provides support for sectioning.

Proper orientation ensures that diagnostic tissue elements are visible during microscopy. Incorrect orientation can lead to damage or loss of critical structures.

Tubular structures – Cross-sections should show the wall and lumen.
Skin biopsies – Cross-sections should include epidermis, dermis, and subcutaneous layers.
Muscle biopsies – Sections should show both transverse and longitudinal planes.

Types include:
Carousel-type processors – Tissues move through stationary reagent containers.
Enclosed vacuum processors – Reagents are moved into and out of a retort chamber using vacuum and pressure.

Microwave processors use heat to accelerate fluid exchange, reducing processing time from hours to minutes.
Advantages: Faster processing, environmentally friendly reagents.
Disadvantages: Labor-intensive, limited tissue size, high equipment cost.

Newer technologies offer:
Faster processing times.
Customizable schedules with vacuum, heat, and agitation.
Environmentally friendly reagents.
Improved standardization and productivity.

Tips include:
Clean spills immediately.
Monitor paraffin wax temperature daily.
Perform warm water flushes to prevent clogging.
Follow manufacturer maintenance schedules.

Common schedules include:
Overnight processing – Uses formalin, alcohols, xylene, and paraffin wax.
Rapid processing – For small biopsies, uses heat and vacuum to reduce time.

Breast tissue – Fixed in 10% NBF for 6–72 hours, then processed through graded alcohols, xylene, and paraffin wax.
Eyes – Fixed in formalin, dehydrated with phenol-alcohol mixtures, and cleared with chloroform.

Small biopsies can be processed in 2–5 hours using heat (37–45°C) and vacuum. Steps include fixation, dehydration, clearing, and infiltration.

Manual processing involves transferring tissues through a series of reagents (fixatives, alcohols, clearing agents, and paraffin wax) manually, without automated equipment.

Dried tissues can be restored using a solution of 70% ethanol, glycerol, and dithionite. The tissue is soaked for several hours or overnight, then processed through dehydration and subsequent steps.

Safety measures include:
Using material safety data sheets (MSDS) for all chemicals.
Wearing protective equipment (gloves, goggles, lab coats).
Properly storing and disposing of flammable and toxic reagents.

Reagents like alcohol and xylene are recycled using distillation equipment. This separates waste products by boiling points, purifying the reagents for reuse while reducing costs and environmental impact.

TMA is a technique that arranges multiple tissue cores in a single paraffin block. It allows simultaneous analysis of hundreds of tissue samples, saving time and resources in research and diagnostics.

Advantages include:
High-throughput analysis of multiple tissues.
Conservation of tissue samples.
Cost-effectiveness for staining and reagent use.
Standardization of immunohistochemistry (IHC) and other assays.

Types include:
Prevalence TMAs – For studying the frequency of specific alterations.
Progression TMAs – For analyzing different stages of a disease.
Prognosis TMAs – For correlating molecular findings with clinical outcomes.
Experimental TMAs – For testing new antibodies or gene targets.

The grid is designed based on the purpose of the array. It includes:
A map or spreadsheet to track core positions.
Control tissues (e.g., normal tissue) placed strategically.
Notches on the block for orientation.

Steps include:
Selecting donor blocks and marking areas of interest.
Punching cores from donor blocks using a needle.
Inserting cores into a recipient paraffin block.
Sectioning the block and staining slides.

Needle sizes include:
0.6 mm – For high-density arrays (up to 400 cores).
1.0 mm – General use (208 cores).
1.5 mm – For fewer cores (99 cores).
2.0 mm – Rarely used due to donor block damage.

A database tracks the position of each core in the TMA block. It allows researchers to correlate staining results with clinical data and analyze multiple cores efficiently.

Types include:
Automated arrayers – High-throughput, with specimen tracking software.
Manual arrayers – Relies on visual identification and manual punching.
Portable quick ray – Economical and easy to use.

Steps include:
Preparing a blank paraffin block as the recipient.
Punching holes in the recipient block.
Extracting cores from donor blocks and inserting them into the recipient block.

The block is sectioned using a microtome to produce 4–5 µm thick sections. Sections are placed on charged slides and stained with H&E or other techniques (e.g., IHC, FISH).

Tips include:
Ensure proper spacing between cores to avoid cracks.
Avoid pushing cores too deep into the recipient block.
Use a dedicated microtome for sectioning.
Seal the block surface with paraffin to prevent antigen loss.

Maintenance includes:
Cleaning residual paraffin from punches and block holders.
Oiling X-Y or Z rails periodically.
Replacing bent or dulled punches.
Avoiding soaking parts in xylene.