Pathology Translational Technology Platform

The Pathology TTP receives human or animal specimens for processing and embedding into wax to create formalin-fixed paraffin-embedded (FFPE) blocks. These must be dissected and fixed for at least 24-48h in 10% NBF or 4% PFA before submission, so that they are ready for processing. We recommend using a fixative volume at least 10 times greater than the tissue volume to ensure thorough fixation.

FFPE wax blocks can also be created from cell pellets, organoids and assembloids that are embedded in agarose.

Very small specimens are processed using a short programme, while larger, bony, or fatty specimens require longer processing times.

After processing, specimens are embedded in cassettes so that the largest and flattest side will be exposed at the cutting surface. Please specify if a different orientation is required

FFPE sections (typically 3-5um thickness) or curls (10um thickness) are cut using a rotary microtome. Researchers need to specify the number of sections required and whether sections should be collected on standard glass slides (for H&E) or coated slides (for IHC). We recommend that slides are baked at 60°C for an hour to ensure the sections are firmly attached.

To enable optimal sectioning, calcium deposits must be removed from bony/calcified samples before processing. The Pathology TTP uses 10% EDTA to decalcify as it preserves tissue morphology and nuclear, antigen and enzyme staining. This produces specimens that are suitable for downstream molecular analyses including immunohistochemistry, in situ hybridisation and PCR. Decalcification is monitored by X-ray until completion. EDTA does inactivate alkaline phosphatase but activity can be restored by the addition of magnesium chloride.

If focal calcium deposits are discovered during sectioning, 10% formic acid is used to soften the block surface.

Frozen sections (typically 5-10um thickness) or curls (10-50um) can be cut from fixed-frozen or fresh-frozen specimens using a cryostat. These are typically collected on coated white slides.

The facility prefers to receive frozen specimens before they are embedded in OCT, so that the sample orientation can be identified. Alternatively, researchers should mark the preferred cutting orientation on prepared frozen blocks. The infection transmission risks of fresh-frozen samples must be discussed with managers before samples are submitted.

The Pathology TTP routinely provides H&E staining to reveal the cellular and tissue morphology. Haematoxylin stains nuclei dark blue-purple while Eosin stains other cellular and extracellular components up to five different shades of pink. This can be performed on FFPE or frozen sections.

The Pathology TTP can optimise other special stains upon request, such as Periodic Acid Schiff, Congo Red and Thioflavin-S. Researchers should get in touch to discuss staining options and controls.

Brightfield, whole slide images can be generated using either the:

• Leica Aperio GT 450 DX scanner -produces images in .svs format at 40x magnification.
• Hamamatsu NanoZoomer S360 produces images in .ndpi format at either 20x or 40x magnification.

Researchers must provide a hard drive onto which the images will be transferred. Images will be retained for up to 30 days.

Upon request, DNA/RNA extractions can be performed on:

• curls cut from FFPE blocks using the Qiagen AllPrep DNA/RNA FFPE kit.
• curls cut from frozen tissues using the Qiagen QIAamp DNA Mini kit.

Please get in touch to discuss the options and timeframes for DNA/RNA extractions.

TMAs are wax blocks that contain small cores of tissue taken from FFPE donor blocks. When sectioned, TMA blocks allow analysis of many samples in parallel.  

Epredia’s automated “TMA GrandMaster” system can produce high-quality TMAs containing up to 500 cores in a fully customisable layout. Alternatively, cores can be collected in PCR tubes for DNA/RNA extraction. 

Whole slide images (in .svs or .tiff formats) can be superimposed and aligned to an image of the FFPE donor block face, to accurately obtain cores from the correct target regions. It also incorporates barcode scanning and block imaging to enable complete sample traceability when mapping cores and blocks.  

Chromogenic Immunohistochemistry (IHC) and In Situ Hybridisation (ISH) can be performed using the Leica Bond MAX or Ventana Benchmark Ultra automated systems. These can be set up to perform dewaxing, epitope retrieval, blocking, antibody incubation and wash steps. They are more cost-effective the greater the number of slides that are run.

Antibodies for immunohistochemistry can be optimised on FFPE or frozen sections upon request. Please get in touch to discuss antibody optimisation and the options for IHC/ISH.

The Pathology and Biobank TTP teams have the necessary permissions to retrieve slides and section wax blocks from the archives on behalf of researchers.

Before accessing this material, researchers must have documented ethical approval in place from the UCL/UCLH Biobank for Health and Disease.

Archival material will be held securely within the facility where Pathology TTP or Biobank TTP staff will scan the slides or section the wax blocks as requested. Archival material will be returned to the Biobank within 3 months of retrieval.

Rotary microtomes are used to cut specimens into sections between 1-60 microns in thickness for detailed microscopic examination.

The facility has several manual rotary microtomes available, as well as cold plates for chilling FFPE blocks and waterbaths for floating out sections.

A chilled microtome for the cutting of frozen sections of 1-100 microns in thickness, at temperatures down to -30°C. It has a quick freeze function for rapid cooling of samples and a UVC disinfection to maintain sterility between samples.

Tissue samples may be fresh or fixed-frozen tissue, and are typically embedded in OCT, agarose or gelatin.

An automated, brightfield slide scanner that combines high image quality (0.26 µm/pixel at 40x) with rapid scanning. It is simple to operate, requires minimal user interaction and up to 450 slides can be loaded at a time.

Slides are automatically scanned at a throughput of 81 slides per hour at 40x for a 15mm x 15mm scanning area, to produce whole slide images in .svs format. Suitable for standard 25x75mm slides only. Images can be viewed using Aperio Imagescope software available from Leica.

Please note: clinical slides must be pseudonymised prior to scanning.

A high-throughput brightfield digital scanner that can handle batches of up to 360 slides at a time to produce digital images in .ndpi format at either 20x or 40x magnification.

Capable of scanning one slide in 30s (at 20× mode for a 15x15mm area). Incorporates slide label anonymisation at point of scanning. Suitable for standard 25x75mm slides only. Images can be viewed in NDP.view2 software available from Hamamatsu.

An automated staining system that is designed to deliver precise and consistent immunohistochemistry and in situ hybridisation assays using Leica’s antigen retrieval and detection systems.

Up to 30 slides (FFPE or frozen) can be loaded at a time, running up to 3 different staining protocols. Ideal for researchers looking to optimise single or dual chromogenic immunohistochemistry staining.

An acoustic focused ultrasonicator, capable of rapid and complete homogenization, tissue disruption and DNA/RNA extraction from a range of sample types and volumes, including FFPE and frozen curls.

Samples can be homogenised in small batches using kits available from Covaris.

Droplet Digital PCR (ddPCR) systems splits PCR reactions into 20,000 droplets. PCR amplification and detection of the template then occurs in each individual droplet, allowing for improved sensitivity to detect low-abundant sequences in a sample.

ddPCR uses reagents and workflows similar to those used for TaqMan probe-based assays. It is particularly useful for precise quantification of gene expression levels, rare DNA targets and copy number variation.

The 2200 TapeStation enables the rapid quantification and QC check of DNA or RNA samples, typically used prior to next-generation sequencing (NGS), microarray and qPCR workflows.

The Qubit® 3.0 Fluorometer accurately measures DNA, RNA, and protein using a highly sensitive fluorogenic dye that emits signals only when bound to specific target molecules. 

The NanoDrop is a full-spectrum, UV-Vis spectrophotometer used to quantify and assess the purity of DNA, RNA and Protein.