Formalin-fixed paraffin-embedded, commonly known as FFPE, is a standard procedure for preserving biological tissue samples. It involves treating fresh tissue with chemicals to halt cellular degradation, then encasing it in a solid block of paraffin wax. FFPE blocks offer a stable and durable format for storing patient biopsies and research specimens for extended periods. This preservation technique is foundational in modern medical diagnostics, particularly in disease identification, and supports scientific investigations. Maintaining tissue integrity over decades makes FFPE an indispensable resource for healthcare and research institutions globally.
The Preservation Process
The creation of an FFPE block begins with fixation, where fresh tissue is immersed in a 10% formalin solution (formaldehyde). Formaldehyde reacts with proteins within the tissue, creating cross-links that stabilize cellular structures and prevent decomposition. This process locks the tissue in a state resembling its living form, arresting decay and maintaining cellular architecture.
Following fixation, the tissue undergoes dehydration, progressively removing water. This is achieved by passing the tissue through increasing concentrations of alcohol, typically ethanol. Removing water is necessary because paraffin wax is not miscible with water, ensuring proper infiltration.
After dehydration, clearing removes the alcohol from the tissue. This involves immersing the tissue in a solvent like xylene, which is miscible with both alcohol and molten paraffin wax. Clearing makes the tissue transparent and prepares it for wax infiltration.
The cleared tissue is then submerged in molten paraffin wax, a process called infiltration. The wax permeates the tissue, replacing the clearing agent and filling cellular spaces. Once infiltrated, the tissue, saturated with molten wax, is placed into a mold, and fresh molten paraffin is poured around it.
As the wax cools and solidifies, it forms a solid paraffin block with the tissue embedded within it. This block provides mechanical support, allowing thin sections to be cut. These sections (2 to 10 micrometers thick) are prepared using a microtome. These tissue slices are floated onto a water bath and mounted onto glass microscope slides for analysis.
Applications in Medicine and Research
FFPE tissue blocks are central to diagnostic pathology, serving as the primary medium for disease identification, especially in oncology. Pathologists examine thin sections from these blocks under a microscope to assess tissue morphology, identify abnormal cell growth, and stage diseases like cancer. The preserved cellular and architectural details allow for precise visual diagnosis, guiding treatment decisions.
These blocks also serve as archival storage for patient samples, often kept for decades. This archiving enables retrospective studies, where researchers revisit past cases to understand disease progression, evaluate treatment outcomes, or discover new correlations. It provides a durable historical record of patient health, allowing for future re-evaluation with emerging technologies.
Beyond diagnostics, FFPE samples are utilized in biomedical research to advance understanding of various diseases. Researchers use these preserved tissues to investigate disease mechanisms, identify biomarkers, and explore the efficacy of therapeutic agents in preclinical studies. Vast collections of archived FFPE samples are a significant resource for large-scale population studies.
The availability of FFPE samples facilitates large-cohort studies, necessary for identifying genetic or protein changes linked to disease susceptibility or progression. This repository supports collaborative research aimed at translating discoveries into improved patient care and innovative treatment strategies.
Information Extracted from FFPE Samples
Histological analysis, typically using Hematoxylin and Eosin (H&E) staining, is a common analysis performed on FFPE tissue sections. Hematoxylin stains cell nuclei blue, while Eosin stains the cytoplasm and extracellular matrix pink, providing a clear visual representation of tissue architecture. This staining technique allows pathologists to identify tissue types, detect abnormalities, and observe cellular changes indicative of disease.
Immunohistochemistry (IHC) is another technique applied to FFPE sections, enabling the detection of specific proteins. This method uses antibodies that bind to target proteins. When the antibody binds, it can be visualized, revealing the presence, location, and abundance of proteins, which helps classify tumors, identify infectious agents, and predict treatment response.
Beyond visual examination, FFPE samples are a source for molecular analysis, allowing extraction of nucleic acids (DNA and RNA) and proteins. While fixation can introduce challenges, advanced protocols allow scientists to isolate these macromolecules for study. Retrieving these molecules from archived samples is transformative for modern research.
Genomic analysis on FFPE-derived DNA includes Polymerase Chain Reaction (PCR) for amplifying specific DNA sequences, and various sequencing methods, including next-generation sequencing (NGS). These approaches identify genetic mutations, gene fusions, and copy number variations that drive diseases like cancer. Studying gene expression from RNA extracted from FFPE tissues provides insights into cellular activity and disease pathways.
Proteomic analysis, though challenging due to protein cross-linking during fixation, is also performed on FFPE samples to study protein expression and modifications. Techniques like mass spectrometry can identify and quantify proteins, offering a complementary view to genetic data. These analytical capabilities underscore the comprehensive information obtainable from FFPE tissues.
Challenges and Advancements
Despite their utility, FFPE samples present challenges, primarily stemming from the formalin fixation process. While formaldehyde preserves tissue morphology, it also modifies and cross-links nucleic acids (DNA and RNA) and proteins. This cross-linking can lead to fragmentation of these molecules, making extraction more difficult.
Chemical alterations during fixation can negatively impact the quality and quantity of extracted DNA and RNA, affecting the accuracy and sensitivity of downstream molecular analyses. For example, fragmented DNA might be less suitable for long-range PCR or certain next-generation sequencing, leading to reduced data yield or biased results. Similarly, protein modifications can interfere with antibody binding in proteomic assays.
Researchers are developing advancements to mitigate these limitations and maximize the utility of FFPE samples. Improved nucleic acid and protein extraction protocols are optimized to reverse cross-links and enhance recovery from fixed tissues. These methods often involve extended incubation times or modified buffer compositions to improve yield and integrity.
Advanced molecular assays are designed to accommodate degraded or modified molecules from FFPE samples. For instance, PCR primers can target shorter amplicon lengths, and sequencing library preparation methods work with fragmented DNA. Innovative preservation methods and fixation alternatives are also explored, aiming to reduce molecular damage while maintaining morphological integrity for future diagnostic and research.