Formalin-Fixed, Parrafin-Embedded (FFPE) tissue
Researchers and clinicians use FFPE tissue blocks extensively in medical research and clinical diagnostics. These blocks preserve tissue structure and cellular details for long-term storage. As a result, they provide a valuable source of archived tissue for reanalysis with molecular techniques.
Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Blocks
FFPE is a popular method for preserving biological samples, especially in pathology, research, and diagnostics. The process involves two key steps:
- Formalin fixation: The tissue is placed in formalin (a formaldehyde solution), which keeps its structure intact and stops decay by stabilizing proteins and nucleic acids.
- Paraffin embedding: After fixation, the tissue undergoes dehydration and is infiltrated with paraffin wax. This makes the tissue easier to store and section for microscopic analysis.
FFPE tissue plays a significant role in histopathology, especially in cancer diagnosis. It allows long-term storage at room temperature, making retrospective studies possible. However, formalin causes cross-links between biomolecules, making it harder to extract and analyze DNA, RNA, and proteins. These issues create challenges for molecular research, such as sequencing and gene expression studies.
Method for Paraffin-Embedding Tissue Blocks
The following standard operating procedure for paraffin-embedding tissue blocks is provided by the Biobank Resource Centre:
Notes On Solid Tissue Procurement
A pathologist should oversee tissue procurement to ensure proper preservation of diagnostic material. Solid tissues, such as tumors, can vary greatly in composition (e.g., containing neoplastic cells, normal cells, and necrosis), so a histological section from each sample should be microscopically examined.
- Avoid cross-contamination, dehydration, and desiccation.
- Keep specimens on wet ice if not processed immediately.
- Handle all human specimens as potential biohazards.
Pre-Analytical Factors Affecting Quality
Several factors can affect the quality of FFPE tissue, impacting its use in research and diagnostics.
1. Fixation Process
- Formalin Type and Concentration: The standard concentration is 4-10% neutral buffered formalin. Changes in concentration can lead to over- or under-fixation, which negatively affects tissue and molecular analysis.
- Under-fixation: Too little time in formalin causes tissue decay and degradation of nucleic acids and proteins.
- Over-fixation: Too much time in formalin causes excessive cross-linking, making it difficult to extract biomolecules.
- Time to Fixation: A delay between tissue excision and fixation allows degradation of DNA, RNA, and proteins, so minimizing this time is essential.
2. Formalin Penetration
- Tissue Thickness: Thick tissue samples can prevent formalin from fully penetrating, leading to uneven fixation and degradation. Tissue samples should be around 4-5 mm thick to ensure proper preservation.
- Formalin Volume: The amount of formalin should be at least ten times the tissue volume to ensure effective diffusion.
3. Paraffin Embedding
- Temperature: Embedding tissue at high temperatures damages proteins and nucleic acids. Embedding at 55-60°C is ideal.
- Paraffin Purity: Impurities in paraffin can interfere with molecular research, so it’s important to use high-purity paraffin.
- Quality of Embedding: Incomplete or poor embedding can leave air bubbles or gaps, which may degrade tissue quality and affect subsequent sectioning and staining.
4. Pre-fixation Factors
- Ischemia: Reduced blood supply before excision causes biochemical changes that degrade molecules. Minimizing time from surgery to fixation helps avoid this issue.
- Autolysis: Delays in processing can lead to rapid tissue breakdown. Quick handling and transfer to fixative are essential.
5. Storage Conditions
- Duration of Storage: Over time, FFPE tissues experience DNA and RNA degradation. While proteins are more stable, they can also degrade over time.
- Temperature and Humidity: FFPE blocks should be stored at stable temperatures and in dry conditions. High humidity can lead to paraffin degradation or the growth of mold, and fluctuating temperatures may cause cracking in paraffin blocks, further damaging the tissue.
- Oxidation: Over time, FFPE tissue can be exposed to environmental factors like oxygen, which may cause oxidative damage, further degrading nucleic acids and proteins.
6. Tissue Type and Condition
- Tissue Composition: Different tissue types respond differently to formalin fixation. For instance, fatty tissues (e.g., adipose tissue) may not fix as well as other types due to the poor penetration of formalin into lipid-rich areas. Similarly, highly vascularized tissues may be more susceptible to autolysis.
- Disease State: Diseased or necrotic tissue can be more difficult to preserve due to existing degradation or poor structural integrity, which may exacerbate the challenges of formalin fixation.
7. Handling and Sectioning
- Microtomy and Sectioning: Improper sectioning (e.g., using dull blades or cutting too thickly) can damage tissue, making it harder to analyze in both histopathology and molecular assays.
- Cross-Contamination: Proper handling during sectioning is crucial to avoid contamination, especially in busy labs.
8. Rehydration and Staining
- Deparaffinization Quality: When rehydrating FFPE tissues for downstream applications (e.g., immunohistochemistry or molecular analysis), incomplete deparaffinization can leave residual paraffin that may interfere with staining and molecular assays.
- Staining Protocols: The quality of histological or immunohistochemical staining can be affected by fixation quality. Poor fixation may lead to weak staining or loss of morphological detail, impacting the diagnostic and research use of the tissue.
9. Extraction Methodology
- Quality of Extraction: The protocols used for extracting DNA, RNA, or protein from FFPE tissues need to be optimized to deal with the challenges posed by formalin cross-linking and paraffin embedding. If extraction protocols are not optimized, the yield and quality of biomolecules may be low, limiting the utility of the tissue in downstream applications.
- Nucleic Acid Fragmentation: FFPE-induced fragmentation of DNA and RNA can vary depending on the fixation conditions. Shorter, more fragmented nucleic acids can be a challenge for molecular techniques like PCR or next-generation sequencing.
10. Artifacts Introduced by Fixation
- Chemical Modifications: Formalin fixation can induce chemical modifications to DNA, such as the formation of methylol adducts or oxidative changes, which can interfere with PCR amplification or sequencing results.
- Protein Cross-linking: Formaldehyde cross-linking can mask protein epitopes, complicating immunohistochemical staining. Heat-induced epitope retrieval (HIER) is often needed but may not fully restore antigenicity for some proteins.
Conclusions
To ensure high-quality FFPE tissue for research and diagnostic purposes, it is important to carefully control pre-analytical factors (such as time to fixation and fixation duration), optimize handling and embedding protocols, and store samples under appropriate conditions. Additionally, validated extraction and analysis methods are necessary to overcome the challenges posed by fixation-induced cross-linking and degradation.
Molecular Studies Using FFPE Tissue
FFPE (Formalin-Fixed Paraffin-Embedded) tissue is a widely used method in biomedical research and clinical diagnostics for preserving biological samples. Researchers originally developed FFPE to maintain tissue structure for histopathology, but it has since become a valuable resource for molecular studies, including DNA, RNA, and protein analysis. Below is an overview of its benefits and challenges:
1. DNA Studies
Researchers frequently use FFPE tissue for DNA extraction and analysis, especially in genomics and cancer research. DNA remains relatively stable under FFPE preservation conditions.
Advantages
- Researchers can archive FFPE samples for many years, allowing for retrospective studies.
- They can also perform whole-genome sequencing and targeted gene panels on FFPE-derived DNA, which helps in studying genetic mutations, copy number variations, and epigenetic changes in diseases like cancer.
- Moreover, PCR-based techniques, such as qPCR, methylation-specific PCR, and next-generation sequencing (NGS), work effectively with DNA extracted from FFPE tissues.
Challenges
- However, the fixation process often causes DNA fragmentation and chemical modifications, like crosslinking with proteins, which complicates analysis.
- To overcome this, researchers need specialized extraction protocols to ensure high-quality DNA and minimize degradation.
2. RNA Studies
Although FFPE tissue works well for DNA studies, analyzing RNA poses more challenges because of its instability and tendency to degrade during fixation.
Advantages:
- Despite these difficulties, researchers have successfully conducted mRNA expression profiling and RNA-seq on RNA extracted from FFPE samples. To address the degradation issue, they modify protocols by using shorter read lengths in RNA-seq.
- MicroRNA analysis is another common application, as microRNAs are smaller and more resistant to degradation compared to mRNAs.
Challenges:
- Unfortunately, formalin fixation usually leads to RNA fragmentation, so only short RNA fragments are retrievable.
- Crosslinking between RNA and proteins further complicates RNA recovery. To obtain usable RNA, researchers often rely on specialized RNA extraction kits and short amplicon protocols.
3. Protein Studies
FFPE tissues are also widely used in proteomics and immunohistochemistry (IHC), which remains one of the most common clinical diagnostic tools.
Advantages:
- Using IHC, researchers can localize proteins within tissue sections, providing crucial spatial information about protein expression.
- Additionally, mass spectrometry-based proteomics has been applied to FFPE samples, although it is still in the early stages of development.
Challenges:
- However, formalin fixation can alter protein structure, making it harder for researchers to detect specific epitopes using techniques like IHC or Western blotting.
- Therefore, researchers often use antigen retrieval methods to reverse the chemical modifications and restore protein antigenicity.
4. Epigenetic Studies
FFPE tissues provide valuable data for studying epigenetic changes, such as DNA methylation and histone modifications.
Advantages:
- Researchers benefit from FFPE tissues when studying archived patient samples, enabling them to explore epigenetic changes related to cancer, as well as developmental biology.
- Furthermore, they can apply techniques like methylation-specific PCR and bisulfite sequencing to DNA from FFPE samples.
Challenges:
- As with other nucleic acid studies, DNA fragmentation caused by fixation presents challenges, so researchers must account for this during extraction and processing.
5. Conclusion
To ensure high-quality FFPE tissue for research and diagnostics, it’s essential to manage pre-analytical factors, optimize processing protocols, and use validated molecular analysis methods. Although challenges remain, FFPE samples continue to provide valuable data for clinical research.
References
Bass BP, Kelly B Engel, Sarah R Greytak, Helen M Moore. A review of preanalytical factors affecting molecular, protein, and morphological analysis of formalin-fixed, paraffin-embedded (FFPE) tissue: how well do you know your FFPE specimen? Arch Pathol Lab Med. 2014 Nov;138(11):1520-30.
Engel KB, Moore HM. Effects of preanalytical variables on the detection of proteins by immunohistochemistry in formalin-fixed, paraffin-embedded tissue. Arch Pathol Lab Med. 2011 May;135(5):537-43.
Hedegaard J, et al. Next-generation sequencing of RNA and DNA isolated from paired fresh-frozen and formalin-fixed paraffin-embedded samples of human cancer and normal tissue. PLoS One. 2014 May 30;9(5):e98187. doi: 10.1371/journal.pone.0098187. PMID: 24878701; PMCID: PMC4039489.
Sadeghipour A., Babaheidarian P. (2019) Making Formalin-Fixed, Paraffin Embedded Blocks. In: Yong W. (eds) Biobanking. Methods in Molecular Biology, vol 1897. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8935-5_22.
Srinivasan M, Sedmak D, Jewell S. Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am J Pathol. 2002 Dec;161(6):1961-71. doi: 10.1016/S0002-9440(10)64472-0. PMID: 12466110; PMCID: PMC1850907.
van Maldegem, et al. Effects of Processing Delay, Formalin Fixation, and Immunohistochemistry on RNA Recovery From Formalin-fixed Paraffin-embedded Tissue Sections. Diagnostic Molecular Pathology 17(1):p 51-58, March 2008. | DOI: 10.1097/PDM.0b013e31814b8866
DNA and RNA Extraction from Formalin-Fixed, Paraffin-Embedded Tissue Biospecimens. NCI Biospecimen Evidence-Based Practices (BEBP).
