Sample requirements | Visium HD

General

For good results with the Visium HD Spatial Transcriptomics pipeline, it is important to make sure that the tissue block (and therefore tissue sections) used for the pipeline are of good quality. Furthermore, proper slicing of the tissue is of importance to the quality of the results. If you are preparing fresh sections from a tissue block to ship to SCD, please read and follow the Guidelines provided by 10x Genomics.

For FFPE samples, follow their Visium HD FFPE Tissue Preparation Handbook, starting from page 43. For Fresh-Frozen (FF) samples, follow their Visium HD Fresh Frozen Tissue Preparation Handbook, starting from page 28.  For Fixed-Frozen (FxF) samples, follow their Visium HD Fixed Frozen Tissue Preparation Handbook from page 33. These handbooks explain the recommended procedure and the right placement onto the glass slides.

The minimum number of tissue sections required are the following:

Sample Quality

The integrity of the RNA and the tissue used should be assessed before proceeding to the Visium HD Spatial Transcriptomics workflow. When using materials with poor RNA or tissue quality the resulting data can be poor, and this cannot be improved bio-informatically. Here we address what QC checks can be done to verify RNA and sample quality. These quality checks can either be done by the client. SCD also offers these checks as a service for a fee. Please speak with our sales team to determine the best setup for your project.

RNA quality

To assess RNA quality from a tissue block, begin with tissue slices from the same sample block used for the actual sample. First, extract the RNA from these mock tissue slices. SCD suggests using the RNeasy FFPE Kit from Qiagen (product number 73504) for this step or RNeasy Mini Kit from Qiagen for FF and FxF samples (product number 74104). Next, run the extracted RNA on a Tapestation RNA High Sensitivity Screentape to evaluate the DV200 percentage. The DV200 % Score, which assesses RNA integrity from tissues, represents the percentage of fragments larger than 200 nucleotides. If the RNA is highly fragmented, it will yield a lower DV200 score. Consequently, a higher DV200 score indicates better quality RNA. For the Visium HD workflow, 10x Genomics recommends using tissues with a DV200 score of over 30%. Although lower scores do not necessarily result in poor data, higher scores are more likely to correlate with greater assay sensitivity.

SCD will only process samples that have a DV200 below 30% if the client accepts the risk of failure. 

When the RNA quality assessment is done by SCD, besides the tissue slices for Visium HD pipeline you will need to provide additional 5-10µm sections in a microtube. For larger tissues (>6.5mm x 6.5 mm) 1 or 2 sections are sufficient. For smaller tissues (<6.5mm x 6.5mm), please provide at least 4 sections to allow for proper RNA quality assessment. 

Morphology Assessment

The standard QC test for Visium HD is to assess RNA quality. However, if the DV200 score falls below 30%, or if you want more assurance of high-quality samples before entering the Visium HD pipeline, a morphology assessment can be performed. To do this, use a mock slice from the same tissue block and perform DAPI/H&E staining to assess the tissue’s morphology and its cells and nuclei. Consult the specific Tissue Prepaation Handbook mentioned before to guide you through the staining procedure. When the DAPI staining is poor, sections of that sample block are unlikely to generate good data. Degraded or poor-quality samples can be identified this way, as the DAPI stain might not bind effectively to the DNA, resulting in weak or uneven staining. Additionally, if the tissue was not properly fixed, poor DAPI staining will occur, as proper fixation is crucial for preserving cellular and nuclear morphology.

By incorporating these steps, you can ensure your samples are of the highest quality before proceeding with the Visium HD workflow.

• SCD provides Morphology Assessment upon request by the client. Additional 10 µm sections need to be provided for the morphology assessment.

Glass slide usage

When you provide freshly sectioned tissue slices to SCD for the Visium HD pipeline, ensure you use a supported glass slide. Failing to do so compromises the sample, leading to tissue detachment during the Visium HD Workflow and resulting in sample loss. Refer to the table below for supported glass slides. Schott Nexterion Slide H – 3D Hydrogel Coated Slides seem to perfrom better when working with difficult samples. If you’re unsure which glass slides to use, contact your SCD representative to verify your options.

*10x Genomics recommends placing tissues with large amounts of connective tissue (like breast, skin, or colon) or composed of multiple cores (TMAs) on Schott Nexterion Slide H – 3D Hydrogel Coated Slides (Schott North America, PN-1800434) to minimize risk of tissue detachment. Schott H slides may also be used if prior detachment has been observed with tissues of interest. Schott Nexterion Slide H slides should be stored at -20°C and equilibrated for 30 min at room temperature prior to use.

Right placement on glass slides

When placing the tissue sections on a glass slide for processing, please follow the Visium HD Tissue Preparation Handbooks as mentioned. The right tissue placement is crucial as wrongly placed tissue cannot be properly transferred to a Visium HD capture slide, and hence analysis of the tissue falling outside the region is not possible. Please see picture below taken from the handbook as reference. 

Regions of Interest

For some tissue types, certain areas of the tissue sections might be of interest. For example, a metastasis element could be residing in a tissue. In this case, it is important to notify SCD that there are certain Regions of Interest (ROIs). You can inform the lab team of SCD by uploading microscopy images of the tissue with explanation in the sample submission form. Please be aware the images shared with SCD can be max 10 MB. By informing SCD about the ROIs, we can make sure that these regions fall within the area that is transferred to the Visium HD capture slide. As the capture areas on these slides are limited (6.5 mm x 6.5mm) and some tissues might exceed this surface area, it is important to align ROIs with the capture areas on the Visium HD slide. 

Shipping

Shipment of tissue sections or blocks to SCD laboratories can be done at 4°C with cold-packs for FFPE samples or with dry ice at -80°C for FF and FxF samples. Try to avoid exposure to direct light to ensure even chilling and preservation of RNA integrity.

When you want to ship FFPE tissue slides, adhere to the following instructions: 

• Dry the tissue slide at 42°C for 3 hours. Afterwards, store the slide in a desiccator at RT for a minimum of 12 hours, but no longer than 2 weeks. 
• When the tissue slides are dried, slides should be shipped in a slide mailer with desiccant pouches. Make sure that the slide mailer is properly closed.
• Finally, ship the tissue slides in the slide mailers at 4°C using cold packs. Secure the slide mailers in the shipment box to avoid damaging the slides.

When you want to ship FF and FxF tissue slides, adhere to the following instructions:

• Transfer slides containing tissue sections to a pre-cooled slide mailer.
• Transfer slide mailer to dry ice.
• If shipping multiple slides, ensure that there is sufficient space in between slides to avoid contact. If placing a slide in the last slot, ensure slide does not face outward.
• Place mailer in a tightly sealed secondary container to limit exposure and keep cold.

Make sure when shipping tissue blocks or sections to properly and clearly label the slides or microtubes to avoid any confusion on sample naming. The sample naming on the slides or tubes should correspond to information uploaded in the sample submission forms. 

When you are ready to ship your samples, do not forget to fill out the sample submission form! This way our team gets the heads up your samples are arriving and will receive all the ins and outs regarding your samples.