Formalin-fixed paraffin-embedded (FFPE) DNA refers to genetic material isolated from tissue samples that have undergone a specific preservation process. This technique involves treating biological tissues with formalin and then embedding them in paraffin wax. FFPE samples represent a significant historical archive, with millions stored globally in biobanks, hospitals, and research centers. These preserved tissues offer a unique opportunity to study diseases and biological processes retrospectively, spanning decades of medical history.
What is FFPE and Why is it Used?
FFPE is a widely used method for preserving tissue samples in histopathology, originating from the early 20th century. The process begins by immersing dissected tissue sections in formalin. Formalin penetrates cells and cross-links proteins, DNA, and RNA, preserving the tissue’s structural integrity. This fixation step is followed by dehydration, clearing, and embedding the tissue in molten paraffin wax.
This preservation method maintains tissue morphology and cellular details over extended periods. Pathologists routinely use FFPE blocks for diagnostic purposes, allowing microscopic examination of tissue to identify various diseases, particularly cancer. The widespread use of FFPE has led to the creation of vast archives of patient samples, serving as a rich resource for clinical and basic research.
Impact of FFPE on DNA
While FFPE preserves tissue structure, formalin fixation chemically damages the DNA within the sample. Formaldehyde induces cross-linking, forming covalent bonds between DNA and proteins. This cross-linking can fragment the DNA into smaller pieces.
Formalin exposure also leads to chemical modifications of the DNA bases. A common modification is deamination, where an amino group is removed from a DNA base. These chemical changes and extensive cross-linking make FFPE DNA more degraded and difficult to work with compared to DNA from fresh or frozen tissue samples. The compromised quality of FFPE DNA necessitates specialized approaches for its extraction and molecular analysis.
Extracting and Analyzing FFPE DNA
Specialized extraction kits and protocols are required to reverse the cross-links and purify the fragmented DNA. These methods commonly involve proteinase K digestion to break down protein-DNA cross-links, followed by heat treatment to melt the paraffin.
After these initial steps, silica membrane-based columns or magnetic silica beads are often employed to purify the DNA. The resulting DNA is typically fragmented, with average fragment sizes ranging from 100 to 500 base pairs, though some methods can yield fragments over 1000 base pairs. For downstream molecular analyses like next-generation sequencing, specialized library preparation techniques are necessary to accommodate the fragmented nature and chemical modifications of FFPE DNA. Techniques such as quantitative PCR (qPCR) are often used to assess DNA integrity and amplifiability before more extensive sequencing.
Applications of FFPE DNA
Despite quality challenges, FFPE DNA holds significant scientific and clinical value. It is important for retrospective studies, allowing researchers to investigate diseases and biological processes using archived samples. This historical perspective is useful for understanding disease progression, identifying long-term biomarkers, and studying rare conditions.
In cancer research, FFPE DNA is widely used to identify genetic mutations and alterations. This enables the discovery of new biomarkers and the development of targeted diagnostic tests. FFPE samples also support infectious disease research by providing access to archived pathogen DNA for epidemiological studies or tracking disease outbreaks. Access to vast biobank collections of FFPE tissues allows for large-scale studies, significantly advancing our understanding of human health and disease.