Question: What best practices should be considered when preparing FFPE samples for the Visium HD Spatial Gene Expression assay?
Answer: 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.
In this article we will provide guidance on the following topics for consideration when preparing and selecting FFPE tissue blocks for processing with the Visium HD Spatial Gene Expression assay:
- Best practices for tissue handling
- Tips for optimal fixation
- Tips for optimal processing
- RNA quality assessment
- RNA fragment size - DV200
- Staining based tissue morphology assessment
Note: The guidance provided in this article is also applicable to FFPE tissue block preparation and selection for the Visium CytAssist Spatial Gene Expression assay.
1. BEST PRACTICES FOR TISSUE HANDLING
Prior to fixation and embedding, tissues should be handled according to the following guidelines to maximize RNA quality and prevent degradation.
Gentle Handling
Tissues should be handled gently to avoid mechanical stress. Mechanical stress may damage tissue structure and prevent proper fixation. Examples of processes that introduce mechanical stress include ischemia, coagulative necrosis from electrocautery, and hemorrhages from surgical trauma.
In the example H&E image above, necrotic regions marked by green lines have signs of nuclear condensation (pyknosis) and nuclear fragmentation (karyorrhexis). These regions are typically characterized by poor RNA quality. The surrounding regions have normal-appearing nuclei.
Minimizing Ischemia/Post Mortem Interval (PMI) and Fixation Timing
Prolonged ischemia and PMI can negatively affect tissue quality. If processing delays occur, store tissues in an isotonic solution and avoid exceeding four hours between tissue resection and fixation, though this time may be tissue dependent. The images below show the negative effect of prolonged ischemia on RNA density.
Delayed tissue fixation may lead to autolysis, degrading tissue and negatively impacting results. Tissue samples should be fixed immediately after resection.
In the example H&E images below, considerable degradation of tissue quality or autolysis occurs as the time from fresh mouse brain tissue collection to fixation increases. In the zoomed in image below, the dentate gyrus region shows considerable deterioration of nuclear architecture (signs of nuclear condensation and nuclear fragmentation) with increasing delays to fix the tissue.
Tissue Storage
Store tissues in a cool and moist environment to prevent tissue drying and degradation. Tissues can be kept in an isotonic solution (e.g. RNase-free PBS) if tissue cannot be fixed immediately. Keeping tissues in isotonic solutions is not suitable for long-term storage. Avoid exceeding four hours. 10x Genomics cannot provide specific guidance on isotonic solutions or RNA preservation products.
2. TIPS FOR OPTIMAL FIXATION
Tissues should be fixed according to the following guidelines to maximize RNA quality and prevent degradation.
Optimizing Fixation Time
Under or over fixation of tissues can negatively impact assay performance. Fixation time may need optimization for specific organisms, tissue types, and disease states. Under fixation of tissues may result in the continued activities of some enzymes after tissue resection, which may cause degradation of proteins, nucleic acids, and lipids. RNA may also not be properly preserved in under-fixed tissues. Lastly, under-fixation can lead to tissue artifacts such as irregular chromatin patterns, overstained cytoplasm (eosin), and common autolysis artifacts such as separation of the epithelium from connective tissue.
Over-fixation may lead to a loss of structural integrity of certain molecular features, lipid oxidation, excessive crosslinking, and decreased antigenicity. Additionally, over-fixation may make sectioning more challenging due to tissue hardening. Over-fixation can lead to tissue artifacts such as irregularly shaped/smaller cells or hyperchromatic nuclei staining.
To avoid under or over-fixation, consider the following:
Tissue Size
The thickness of the tissue significantly affects fixative penetration. Freshly collected tissues should be trimmed with a scalpel to ensure that the thickness of the tissue piece is not larger than a standard tissue processing cassette used for preparation of FFPE blocks. Typically, a tissue thickness of 5 - 10 mm would be accommodated by most standard tissue cassettes and ensure proper penetration of fixative.
Fixative and Fixative to Tissue Ratio
The use of 10% Neutral Buffered Formalin (NBF) at a fixative to tissue ratio of 20:1 is recommended for optimal performance.
Fixation Time and Temperature
Fixation should be carried out at 4°C for ~16-24 h to ensure proper fixative penetration. Fixation time may be tissue-dependent and require optimization. Ensure tissue is completely submerged and introduce light agitation as shown in the image below.
3. TIPS FOR OPTIMAL PROCESSING
Tissues should be embedded according to the following guidelines to maximize RNA quality and prevent degradation. These steps generate a formalin fixed & paraffin embedded block that is ready for sectioning.
i) Dehydration
After fixation, tissues are dehydrated in an ethanol series to displace any water remaining in the tissue. Inadequate dehydration may result in poor preservation of tissue structure and cause damage during sectioning.
ii) Clearing
Tissues are "cleared" using a solvent to remove any remaining ethanol. Inadequate clearing may lead to poor wax infiltration, which can result in hard and brittle blocks that are difficult to section.
iii) Infiltration
After clearing, wax is applied to the tissue and allowed to infiltrate. Inadequate wax infiltration may result in holes or gaps in the final block, which may lead to sectioning difficulties. Typically, wax is melted at 60–62°C prior to adding to the tissue. It is then allowed to cool to 20°C. Avoid excessive heat and check temperatures regularly during embedding. Use a high-quality wax with a low melting temperature.
iv) Embedding
Finally, tissues are embedded in paraffin. Record orientation of the tissue during paraffin embedding to ensure proper spatiality for downstream region selection.
Additional Tips and Best Practices
- Regularly replace reagents used during the fixation and embedding process.
- DO NOT store or leave samples in fixative for an extended period, which will lead to over fixation.
- Proceed immediately from fixation to dehydration, clearing, and embedding. If proceeding to dehydration immediately is not possible, transfer tissue from fixative to 70% ethanol for short-term storage.
- Store FFPE blocks properly to maintain integrity. Avoid exposure to high temperatures or humidity. For optimal storage, store at 4°C.
4. RNA QUALITY ASSESSMENT
Assess tissue block RNA quality by calculating DV200 of freshly collected FFPE tissue sections. Visium HD FFPE Tissue preparation Handbook (CG000684) provides guidance on determining the RNA quality of the tissue block by calculating DV200 of RNA extracted from freshly collected FFPE tissue sections.
If extra sections are available, placing them on slides for Optional Tissue Morphology Assessment is recommended. Use the decision tree below to determine what quality assessment protocols should be followed.
Optional Tissue Morphology Assessment
Assessment of tissue morphology prior to performing the Visium HD assay is recommended, but not mandatory. This assessment is composed of DAPI and H&E staining. After staining, tissues are assessed to determine suitability for the Visium HD assay.
DAPI and H&E may be performed on the same tissue section or on serial sections. If extra tissue sections are not available to perform this assessment, H&E or IF images generated during the HD workflow may be evaluated to gain additional insights on sample quality.
Optional DAPI Staining for Tissue Morphology Assessment
If extra tissue slides are available, optional tissue morphology assessment via DAPI and H&E staining is recommended.
DAPI Staining
- Place the tissue slide on a flat, clean, non-absorbent work surface.
- Add 500 µl DAPI working solution (stock - 1mg/ml solution; working - 0.005mg/ml ) per slide to uniformly cover all tissue sections.
- Incubate 1 min in the dark at room temperature.
- Discard reagent by holding slides at an angle with the bottom edge in contact with a laboratory wipe
- Add 1 ml 1X PBS per slide to uniformly cover all tissue sections.
- Incubate 1 min in the dark at room temperature.
- Discard reagent by holding slides at an angle with the bottom edge in contact with a laboratory wipe
- Repeat steps e-g twice more for a total of three washes.
- Mount coverslip as described in 4.6 Coverslip Mounting on page 78 and image as described in Step 4.7 Imaging on page 78 of the Visium HD FFPE Tissue Preparation Handbook (CG000684).
Quality Assessment
Review the DAPI image under the microscope at 20x magnification. Examine several sections within a single DAPI-stained section to ensure assessment is comprehensive. Tissue sections optimal for the assay should have:
- Punctate nuclei that are clearly defined
- DAPI staining that does not appear washed out
Sections with poor DAPI staining are unlikely to generate good data. All samples in the images below were imaged under the same conditions (405 nm channel). After evaluating DAPI staining, remove coverslip as described in 4.8 Coverslip Removal on page 79 of the Visium HD FFPE Tissue Preparation Handbook (CG000684) and proceed to Optional H&E Staining for Tissue Morphology Assessment.
Optional H&E Staining for Tissue Morphology Assessment
H&E Staining
Perform H&E staining as described in 3.2 H&E Staining on page 50, coverslip as described in 3.3 Coverslip Mounting on page 51 and image as described in 3.4 Imaging on page 52 of the Visium HD FFPE Tissue Preparation Handbook (CG000684).
Quality Assessment
Ensure that the tissue section is free from tissue processing artifacts such as necrosis, large cracks, and hemorrhaging. If imaging reveals satisfactory tissue morphology, proceed with the appropriate staining protocol using a different tissue slide. Refer to the Visium HD FFPE Tissue Preparation Handbook (CG000684) for the desired staining protocol.
Staining protocols:
- H&E Staining & Imaging on page 42
- IF Staining & Imaging on page 57
Relevant links:
- Visium HD FFPE Tissue Preparation Handbook (CG000684)
- Visium HD Spatial Applications Imaging Guidelines (CG000688)
Product: Visium HD