Question: Do you have recommendations for FFPE tissue fixation 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.
Poor transcript density on Xenium can be related to many factors including the tissue to panel match, sample biology (e.g., presence of blood, necrotic tissue), workflow errors, etc. Reduced decoding performance can also be related to the input FFPE sample itself. In-house, we have observed that improper sample fixation (over- or under-fixation) can lead to poor transcript density, either across the entire tissue sample or in the interior of the sample. It can also lead to nuclei degradation, which negatively affects segmentation and transcript assignment to cells.
Potential Impact of Under-fixation
Under-fixation can lead to the continued activity of certain enzymes post-mortem or post-operation, which can contribute to the degradation of proteins, nucleic acids, and lipids. Tissues may not adequately preserve the RNA if under-fixed, leading to fragmented strands of nucleotides. Under-fixation can lead to the presence of artifacts in the tissue such as irregular chromatin patterns, overstained cytoplasm (eosin), and common autolysis artifacts (such as separation of epithelium from connective tissue)
Potential Impacts of Over-fixation
Over-fixation can lead to the loss of structural integrity of certain molecular features, the oxidation of lipids, excessive cross-linking, and decreased antigenicity. Tissue hardening is a sign of over-fixation, which may make sectioning difficult. Over-fixation can lead to the presence of artifacts in the tissue such as irregularly shaped or smaller cells or hyperchromatic nuclei staining.
Tips and Tricks for Sample Processing for Optimal Fixation
Here we provide tips and tricks for sample processing to avoid over- or under-fixation. The following factors contribute to, but are not the sole determinants of, fixation success:
1. Tissue size
2. Fixation time and temperature
3. Fixative and the fixative-to-tissue ratio
Additional factors (include the sample type) impact fixation so the guidance provided here may require further optimization.
Formalin penetration is crucial for preserving tissue morphology and molecular integrity, which directly impacts the quality of RNA obtained from FFPE samples.
The thickness of the tissue sample significantly affects formalin penetration.
- In general, tissue sections should be no larger than a standard tissue cassette (example here).
- Thick sections will not allow for adequate penetration of fixative. Inadequate formalin penetration, due to large tissue size, can lead to uneven fixation, resulting in areas of the tissue being under-fixed or over-fixed, which can affect the quality of RNA.
Fixation Time and Temperature
- The fixation process should be carried out at 4°C for ~16-24 hrs to ensure proper penetration and fixation. Fixative penetration rates depend on tissue thickness, with approximate time of 1mm per hour.
- Ensure tissue is submerged and introduce light agitation.
Fixative and the fixative to tissue ratio
- The use of 10% Neutral Buffered Formalin (NBF) is recommended for fixation as it provides a balance between tissue preservation and molecular integrity.
- Ratio of fixative to tissue: 20:1.
Lessons Learned at 10x
This is a simplified representation of sample collection and block preparation:
- Fixation: Having too large tissue sections or too little fixation can lead to under-fixation. Leaving the sample too long in fixative can lead to over fixation. DO NOT store samples in fixative.
- Sample handling delays: For optimal assay performance, it is important to proceed quickly from sample collection to fixation to dehydration/clearing to embedding. Samples should not be stored long-term in PBS at any stage in the workflow. If it is not possible to proceed immediately to dehydration and clearing, a sample can be transferred from fixative to 70% ethanol for short-term storage.
- Dehydration: This step is necessary because paraffin wax, which is used in the embedding process, is hydrophobic (immiscible with water). Therefore, most of the water in a specimen must be removed before it can be infiltrated with wax. This is commonly achieved by immersing specimens in a series of ethanol (alcohol) solutions of increasing concentration until pure, water-free alcohol is reached. If the tissue is not adequately dehydrated, water may remain in the tissue, which can prevent the effective infiltration of the paraffin wax. This can lead to poor preservation of the tissue structure and potential damage during sectioning.
- Clearing: Following dehydration, an intermediate solvent that is fully miscible with both ethanol and paraffin wax is used to displace the ethanol in the tissue. This solvent (in-house we utilize Xylene or Neo-Clear), in turn, is displaced by molten paraffin wax. This stage in the process is called “clearing.” The clearing step can be compromised if not done correctly. Incomplete clearing can result in poor wax infiltration, leading to hard and brittle blocks that are hard to section.
- Wax Infiltration: After clearing, the tissue can be infiltrated with a suitable histological wax (in-house we utilize Leica ParaPlast). A typical wax is molten at 60°-62°C and can be infiltrated into tissue at this temperature. It is then allowed to cool to 20°C. If the wax does not infiltrate the tissue, it can result in holes or gaps in the final block, which can cause problems during sectioning and negatively impact Xenium results.
- Paraffin Embedding: Now that the biospecimen is thoroughly infiltrated with wax, it must be formed into a “block” which can be placed into a microtome for sectioning. This step is carried out using an embedding station (in-house we utilize Epredia HistoStar) where a mold is filled with molten wax and the specimen placed into it. Upon placement, mold is left on a cold plate to solidify. Record orientation of tissue in the block during embedding to ensure proper spatiality for downstream region selection and analysis.
- Inadequate formalin penetration due to tissue size or insufficient fixative can lead to uneven fixation, resulting in areas of the tissue being under- or over-fixed, which can affect the quality of RNA.
- Penetration rates for formalin are around 1 mm per hour and decrease with depth.
- Formalin fixation results in cross-linking of proteins and nucleic acids, which can lead to degradation of RNA if not properly managed.
- The fixation process should be carefully controlled to minimize degradation.
- If tissue cannot be trimmed and is too thick, a longer time in fixation may be needed. In such cases, best to replenish the fixative every 48 hrs.
Product: Xenium In Situ Gene Expression