Formalin-Fixed Paraffin-Embedded (FFPE) tissue processing is a widely used method for preserving biological samples. This technique involves treating fresh tissue with formalin, a formaldehyde solution, and then embedding it in paraffin wax. The primary goal of FFPE is to maintain the structural integrity of tissues, allowing for long-term storage and detailed examination.
Importance of FFPE in Tissue Analysis
FFPE tissue plays a significant role in clinical diagnostics and scientific research by preserving tissue morphology and cellular details. This preservation allows for thorough microscopic examination, which is valuable in diagnostic pathology. It is a standard procedure for diagnosing a wide array of diseases, including various types of cancer, by enabling pathologists to observe cellular abnormalities and tissue architecture.
The method also enables the long-term archiving of tissue samples for research. FFPE blocks remain stable for years, even decades, at room temperature, making them a resource for retrospective studies. Researchers can revisit these archived samples to explore disease progression, evaluate treatment responses, and identify potential biomarkers.
Key Stages of FFPE Processing
FFPE processing begins with the collection of fresh tissue, often obtained through biopsies or surgical resections. This initial step requires careful handling to prevent degradation of cellular components, ideally involving immediate transfer to a fixative solution. Tissue size is also a consideration for optimal processing.
The next stage is fixation, where the tissue is immersed in a liquid fixing agent, most commonly a 10% neutral buffered formalin solution. Formalin works by cross-linking proteins, DNA, and RNA, halting cellular processes and preserving the tissue’s structural and molecular components. Fixation duration typically ranges from 6 to 24 hours, depending on tissue type and size, to ensure thorough penetration and preservation.
Following fixation, the tissue undergoes dehydration to remove water, as paraffin wax is not soluble in aqueous solutions. This is achieved by immersing the tissue in a graded series of ethanol solutions, progressively increasing to 100% ethanol. This gradual process prevents rapid degradation of the tissue and proteins. Improper dehydration can result in incomplete saturation of the sample with paraffin wax and softness of the tissue.
After dehydration, a clearing agent, such as xylene, is used to remove ethanol from the tissue. Xylene also helps remove unnecessary fat, making it easier for paraffin wax to penetrate the tissue and giving it a transparent appearance.
The cleared tissue is then infiltrated with molten paraffin wax. This involves placing the tissue in liquid paraffin, allowing the wax to permeate thoroughly. The tissue is then embedded in a mold with fresh liquid paraffin, which solidifies to form a solid block. Proper orientation of the tissue within this paraffin block is important as it determines the plane of sectioning for later microscopic examination.
The final stage of FFPE processing is sectioning, where thin slices are cut from the solidified paraffin block. A specialized instrument called a microtome is used to cut these sections. These thin sections are then floated on a warm water bath to remove wrinkles before being picked up onto glass slides, making them suitable for staining and microscopic viewing.
Challenges and Limitations of FFPE
While FFPE processing is effective for preserving tissue morphology, it presents challenges, especially concerning molecular analysis. Formalin fixation, which involves cross-linking of proteins, can lead to protein denaturation. This alteration in protein structure can affect the performance of some downstream analyses, such as immunohistochemistry assays, where antibody binding might be compromised.
A limitation of FFPE is the potential for damage to nucleic acids, particularly RNA, due to the formalin fixation process and long-term storage. Formalin can cause fragmentation and chemical modifications of DNA and RNA, making it more difficult to extract high-quality molecules for molecular studies. RNA is generally less stable than DNA and is more susceptible to degradation during FFPE preparation, impacting the feasibility of RNA-based analyses.
The FFPE process can introduce artifacts such as tissue shrinkage or cracking, which might obscure interpretation during microscopic examination. Variations in processing protocols can also influence the quality of the FFPE sample and its suitability for subsequent analysis.