Paraffin Tissue Processing

Paraffin tissue processing plays a fundamental role in histopathology, which involves the microscopic examination of tissues and cells to diagnose diseases.

Paraffin tissue processing prepares biological tissue for thin sectioning (slicing) and microscopic examination. Microscopy demands ultra-thin sections of tissue (typically 4 - 10 micrometres thick). Fresh tissue, which is soft and fragile, requires the support and rigidity provided by paraffin wax to secure consistent, high-quality standards.

Scientists value this technique for its ability to preserve specimens perfectly. The process works with paraffin wax that melts between 56-58°C and contains straight chains of 20-40 carbon atoms - ideal conditions for preserving tissue.

The technique involves several key steps:

Fixation: This entails preventing degradation by preserving the structure of the tissue.
Dehydration: Water is removed from the tissue by increasing concentrations of alcohol, ethanol or isopropanol (e.g.,50%, 70%, 95%, and 100%).
Clearing: Samples are immersed in xylene, a miscible solvent with the dehydrating agent and paraffin.
Infiltration: Molten paraffin wax is injected into the tissue.
Embedding: The infiltrated tissue is then encased in a block of solid paraffin wax.

These prepared samples, called FFPE (formaldehyde-fixed, paraffin-embedded) tissues, are preserved indefinitely for routine morphological staining.

This article will introduce you to the science of paraffin tissue embedding. The content will be helpful to both newcomers to histology and experienced professionals who want to enhance their tissue-processing skills. These insights will help you achieve the best results.

Why Paraffin?

Paraffin is the perfect medium for embedding tissue because of the following properties:

Uniformity: It provides a relatively uniform and consistent support medium.
Sectioning: This allows for the production of thin, serial sections.
Staining: Paraffin is easily removed from the tissue sections, allowing for staining with various dyes to visualise cellular structures.
Storage: Paraffin blocks can be stored for long periods
Cost-Effective: Paraffin blocks also cost just 10% of alternative mediums like historesin.

Fundamentals of Paraffin Tissue Embedding

Paraffin wax forms the foundation of histological preparations. Its chemical structure consists of straight-chain hydrocarbons with 20-40 carbon atoms. This versatile material melts between 56-58°C, which creates perfect conditions for tissue infiltration before it hardens at room temperature.

Tissue embedding starts after proper fixation and dehydration. Paraffin's insolubility in water means specimens need complete dehydration through increasing alcohol concentrations (70%, 90%, and 100%). The process requires clearing agents like xylene that mix with both alcohol and paraffin. This vital step makes tissues translucent and ready for complete paraffin infiltration.

Today's paraffin formulations contain plastic polymers sold under brands like Paraplast and Paramat. These new formulations give better sectioning quality and tissue support. Paraplast Plus contains dimethyl sulfoxide (DMSO) that speeds up infiltration and enables thinner sectioning down to 2-4μm.

Paraffin offers several advantages over other embedding media. FFPE tissues maintain excellent morphological preservation and can remain at room temperature for years, while frozen tissue needs constant ultra-low temperatures.

The process does have some drawbacks:

Tissues can shrink up to 15-20%, which might affect morphological measurements.
The fixation and infiltration process can also fragment DNA and cross-link proteins, affecting some molecular analyses.

Despite these limitations, paraffin is the top choice for routine histopathology. Embedded tissues can be cut with a microtome into thin 4-6μm slices that maintain excellent morphological detail for diagnostic and research purposes.

Critical Considerations for Embedding

Embedding is a crucial step in paraffin tissue processing that significantly impacts the quality of downstream analyses. Proper orientation, block stability, and the minimisation of artefacts to ensure optimal sectioning and accurate diagnostic interpretation are essential for producing high-quality slides suitable for microscopic examination.

Let’s highlight a few critical considerations during embedding:

Orientation: Proper tissue orientation is essential. How the tissue is placed in the paraffin block determines the plane of section and the structures that will be visible under the microscope.
Paraffin Quality: It is essential to use high-quality paraffin wax with the correct melting point (usually around 56-58°C). Too high a melting point can damage the tissue.
Speed: Once the tissue is out of the infiltration step, you need to work quickly to prevent the paraffin from solidifying too much before the tissue is oriented correctly.
Bubble Avoidance: Minimise air bubbles around the tissue during embedding, as they can cause problems during sectioning.
Cooling: Controlled cooling is important to prevent tissue distortion.

Step-by-Step Paraffin Processing Workflow

High-quality histological specimens demand careful attention for standardised tissue processing. The process takes tissue from a fixed state to an embedded paraffin block through three vital stages:

Dehydration: This involves removing water by soaking tissue in various ethanol solutions. Tissue cassettes are sequentially immersed in increasing concentrations of water. Complete dehydration is vital because paraffin wax and water do not mix. The τ90 (time to 90% completion) varies for different concentrations - 70% ethanol needs 2.86 to 7.25 hours, 90% ethanol takes 1.17 to 4.10 hours, and 100% ethanol requires 0.97 to 5.87 hours. Poor dehydration creates brittle sections due to inadequate paraffin infiltration.
Clearing: Xylene is a common clearing agent. It replaces the alcohol with a solvent that mixes with alcohol and paraffin wax and clears tissues, making them transparent and ready for paraffin infiltration with τ90 ranging from 1.06 to 2.14 hours. Xylene is more accessible, but it poses health risks like respiratory irritation, dizziness, and potential nervous system damage. Limonene, UltraClear™, and cedarwood oil offer safer options.
Infiltration: Molten paraffin wax (typically 56-58°C) replaces the clearing agent, thoroughly impregnating the tissue. Multiple paraffin baths ensure that clearing agents are fully displaced. Vacuum application substantially improves infiltration effectiveness. Three changes of paraffin yield the best results.

Temperature control plays a key role throughout this process. Gentle heat (2-4°C above paraffin's melting point) helps infiltration, but excess heat can harden tissue. The process needs careful monitoring. Ultrasound time-of-flight (TOF) technology can detect processing errors by tracking fluid exchange rates.

The completed infiltration allows tissue transfer to the embedding stations. Technicians orient tissues in metal moulds, cover them with molten paraffin, and let them solidify. This creates paraffin blocks ready for microtome sectioning, producing thin tissue slices for microscopic examination.

Take-away

Modern histopathology laboratories rely heavily on paraffin tissue processing. This technique preserves tissue samples reliably through dehydration, clearing, and infiltration steps. The method serves both diagnostic needs and research applications effectively.

The process works best with precise temperature control and proper chemical handling. Each processing stage requires careful attention to detail. Paraffin remains the top choice for routine histological work because it is economical, stores well long-term, and perfectly preserves morphological features.

However, paraffin processing does have limitations—tissue can shrink, and molecular analysis might face challenges. Yet the method keeps getting better with improved formulations and newer methods. This proven approach helps researchers better understand tissue structure and disease processes.