Formalin-Fixed, Paraffin-Embedded (FFPE) tissue is the universal method for preserving tumor samples removed during biopsies or surgery. Developed in the late 19th century, this technique halts cellular decay and creates a stable physical block of the specimen. The FFPE block is foundational to modern anatomical pathology, serving as the definitive record of a patient’s disease for initial diagnosis and subsequent molecular analysis. This practice ensures the tissue is preserved indefinitely for diagnostic confirmation, research, and new treatment strategies.
The Preservation Process
The journey of a fresh tumor sample into an FFPE block involves a precise, multi-step process designed to maintain cellular architecture. The first step is fixation, typically using a 10% neutral-buffered formalin solution. Formalin chemically stabilizes the tissue by forming cross-links between proteins, creating a molecular mesh that prevents decomposition and preserves cellular structure.
Following fixation, the tissue undergoes dehydration and clearing to prepare it for wax infiltration. Water in the tissue is gradually replaced with increasing concentrations of ethanol, and then a clearing agent, such as xylene, is used to remove the alcohol. The clearing agent allows the tissue to fully accept the molten paraffin wax. Finally, the tissue is infiltrated with hot paraffin wax, which solidifies upon cooling to produce a durable block suitable for long-term storage.
Role in Standard Cancer Diagnosis
The traditional use of the FFPE block is to provide a physical specimen for visual examination, accomplished through microtomy. This process uses a specialized instrument called a microtome to cut extremely thin sections, typically between 3 to 5 micrometers thick, from the paraffin block. These tissue sections are then mounted onto glass slides for staining and microscopic assessment by a pathologist.
The mounted tissue is subjected to the Hematoxylin and Eosin (H&E) stain, the standard procedure for initial cancer diagnosis. Hematoxylin stains cell nuclei blue-purple, while eosin stains proteins pink. This differential staining allows the pathologist to visualize cellular morphology, including nuclear size, cell shape, and overall tissue architecture. Through this morphological analysis, the pathologist classifies the tumor type, assigns a histological grade, and assesses surgical parameters, such as the completeness of tumor removal.
Extracting Molecular Data
Beyond structural analysis, FFPE blocks are sources for modern molecular diagnostics that inform targeted treatments. Despite chemical damage caused by formalin fixation, specialized laboratory protocols can reverse the cross-links and extract nucleic acids (DNA and RNA) and proteins. Retrieving these molecules allows for a deeper understanding of the tumor’s underlying biology.
Extracted DNA and RNA are used in targeted gene sequencing to identify specific mutations, gene fusions, or amplifications that can be therapeutically targeted. For instance, identifying an EGFR mutation in a lung tumor can direct a patient toward a specific inhibitor drug. Proteins are analyzed using Immunohistochemistry (IHC), which uses antibodies to visualize the presence and location of specific biomarkers, such as hormone receptors or immune checkpoint proteins like PD-L1, directly on the tissue section. Fluorescence In Situ Hybridization (FISH) uses fluorescent probes to detect specific DNA sequences or gene rearrangements, such as HER2 amplification, which guides treatment decisions.
The Value of Tissue Biobanks
The long-term preservation capability of FFPE gives significant value to tissue biobanks. Hospitals and research centers maintain vast archives of these blocks, creating a historical library of patient disease spanning many decades. Each block is a permanent physical record of the disease at the time of diagnosis, often linked to extensive clinical data regarding patient outcomes and treatment responses.
These biobanks enable researchers to perform retrospective studies, testing new diagnostic or prognostic markers against samples collected years or decades ago. A newly discovered cancer biomarker can be validated against thousands of archived specimens, correlating its presence with long-term survival data. This archival resource is beneficial for studying rare cancers, where gathering a large, contemporary patient cohort would be impractical.
Challenges in Analyzing Fixed Tissue
While FFPE samples are valuable for their abundance and long-term storage, the fixation process introduces technical hurdles for molecular analysis. Formalin chemically modifies and fragments nucleic acids, making the extracted DNA and RNA significantly degraded compared to fresh-frozen tissue. This degradation complicates sequencing efforts, as fragmented molecules are difficult to amplify reliably for downstream applications.
The protein cross-linking, necessary for structural preservation, also affects molecular integrity and yield, reducing protein accessibility for some assays. Scientists must employ specialized extraction kits and optimized protocols that include steps to reverse the formalin-induced cross-links before successful sequencing or proteomic analysis. Advancements in sequencing technology continue to improve the ability to recover meaningful data from degraded FFPE specimens.