Question: Do you have tips and tricks for pre-fixation tissue handling for Xenium?
Answer: These tips and tricks have been developed by the Xenium sample preparation team and applies to the Xenium product family only. The guidance provided here is not officially supported and provides general recommendations based on literature searches and lessons learned. 10x Genomics cannot provide additional information outside what is stated in this article, and any information provided here should be considered a starting point for further optimization depending on the experimental design and tissue type.
Formalin-fixed paraffin-embedded (FFPE) samples are a standard in histopathology labs for diagnosis, research, and teaching and can be used for a variety of diagnostic procedures, including in situ hybridization, which allow for the visualization of specific nucleic acids within a tissue sample of interest. FFPE samples are a supported input sample type for Xenium In Situ Gene Expression (Xenium In Situ Gene Expression - FFPE Tissue Preparation Guide).
The process of FFPE tissue preparation starts with fresh tissue, which is then fixed with 10% neutral buffered formalin (NBF) or 4% paraformaldehyde (PFA). The tissue is then embedded in paraffin wax to provide a firm medium for sectioning and long-term storage stability. However, the process of fixation and embedding can adversely affect the quality of analytes, particularly RNA, obtained from such archived specimens. RNA is particularly susceptible to degradation, and maintaining its quality in FFPE blocks can be challenging.
The following factors can help maintain RNA quality and tissue integrity prior to fixation:
1. Gentle handling of fresh tissue
2. Minimizing post-mortem intervals
3. RNAse-free, isotonic storage conditions prior to fixation
4. Immediate fixation of fresh samples
Best Practices for Fresh Tissue Handling:
Fresh tissue samples should be handled with care to prevent any physical damage. Any mechanical stress or damage can affect the tissue structure and potentially influence the fixation process1. For example, ruptured cells due to ischemic differences, coagulative necrosis from procedures using electrocautery, and hemorrhages from surgical trauma.
The Importance of Ischemia Time and Post-Mortem Interval (PMI):
Ischemia time and PMI are crucial factors that can affect the quality of the tissue sample. Prolonged ischemia time can lead to tissue degradation and affect the quality of the results. Therefore, it is important to minimize the time between tissue resection and fixation (no more than 4 hrs as best practice, depending on tissue type1).
Storage Conditions Prior to Fixation:
The conditions under which the tissue samples are stored before fixation can also impact the quality of the fixed tissue. It is recommended to store the tissue samples in a cool and moist environment to prevent drying out and degradation2. If the sample cannot be fixed immediately, an isotonic solution/saline (for example RNase-free PBS) may be used to maintain tissue hydration and ionic balance. This said, isotonic solutions are not suitable for long-term storage (and it is a best practice to proceed immediately to fixation). We have not tested storage solutions such as PaxGene3 or RNAlater in-house. We also cannot provide recommendations for isotonic solutions for storage that might be compatible with Xenium.
Timing of Fixation:
The timing of fixation is important as described above. Delayed fixation can lead to autolysis, which can degrade the tissue and affect the quality of the results. Therefore, tissue samples should be fixed immediately after resection/excision2.
The considerations outlined above are general recommendations for specimen processing prior to fixation. Additional best practices can be found in ‘An Introduction to Specimen Processing’4.
- PlosOne: "The Influence of Tissue Ischemia Time on RNA Integrity and Patient-Derived Xenografts (PDX) Engraftment Rate in a Non-Small Cell Lung Cancer (NSCLC) Biobank"
- J Mol Sci: "Pre-Analytical Considerations for Successful Next-Generation Sequencing (NGS): Challenges and Opportunities for Formalin-Fixed and Paraffin-Embedded Tumor Tissue (FFPE) Samples"
- Virchows Archiv: "Histological evaluation of PAXgene tissue fixation in Barrett’s esophagus and esophageal adenocarcinoma diagnostics"
- Leica: “An Introduction to Specimen Processing”
Product: Xenium In Situ Gene Expression