Slidepath

The Digital Morphology CPD scheme has been described in earlier publications [14 (link)]. In brief, more than 50 continuous microscopic fields were captured using Zeiss Axio Imager.M1 with a Zeiss Plan-Apochromat 63×/1.4 oil lens, then stitched using the photomerge function of Adobe Photoshop CS4 (version 11.02; Adobe Inc) and optimized for sharpness (unsharp mask function, 1 pixel up to 100%), lighting, and color balance (curves function). The final image was reviewed to ensure accurate and sufficient representation of the diagnostic features for each case, then uploaded to either Digital Slidebox (SlidePath, Leica Biosystems) or EQATE UK NEQAS Haematology Online (Certus Technology Associates Ltd). Each system provided equivalent functionality for image viewing and data submission (representative images from the EQATE system are shown in Multimedia Appendix 1). Users of the UK NEQAS DM scheme viewed the images via web browser, submitting their morphological selections and preferred diagnosis. Feedback was made using online forms with free-text responses or user grading (1-5; Google Forms, Google). The 5 cases that were rereleased used identical films. The cases were given new narratives and feedback available after case completion, and participants had no direct access to the previous release of these cases.

Murine tissues were OCT embedded and frozen in chilled isopentane using liquid nitrogen. Cryopreserved tissues were then sectioned at 10 μm on a cryostat (model CM1850, Leica Biosystems). The PAS/D-PAS, H&E, and staining on frozen tissues followed the methods published in Dubowitz et al.⁶⁹ One whole forelimb and hindlimb per mouse were instead fixed in formalin, skinned, and inked to identify the dorsal/ventral orientation, decalcified, and paraffin embedded. Sections at 4 μm were cut through the limb, proximal to distal (shoulder to paw) and stained for H&E. All stained slides were scanned and captured by Leica SCN400, while images were viewed and captured by using the Leica Slidepath digital image hub application.

Tissue were fixed in situ with 4% paraformaldehyde (Sigma), stored in 70% ethanol (4 °C) and were then processed through a series of graded ethanol, from 70% to 100%, Xylene, and paraffin (Leica Peloris). Heart and vessels (n = 6/group) were sectioned at 5 µm onto charged slides using a rotary microtome as previously described^{18 (link),45 (link)}. Sections were stained with hematoxylin/eosin for primary assessment of structural architecture and Gomori Trichrome for assessment of collagen deposition. The stained slides were then digitally scanned (Slidepath, Leica). Investigators were blinded to experimental condition during selection and measurement of all fields^{16 (link),18 (link),45 (link)}. Immunohistochemistry (Nikon Eclipse fluorescence upright microscope and processed with NIS Elements Basic Research Microscope Imaging Software) of 5-µm sections of mouse heart and lung was performed using 1°-antibodies, anti-α-SMA (1:200), corresponding 2°-antibodies (1:250) and DAPI (1:10,000) (SantaCruz) performed (Olympus-BX51/NikonD5100).

Nerve biopsies were processed according to local standard operating procedures. In brief, fresh biopsies, immediately after removal, were divided in half, with one portion fixed in 10% buffered formalin, followed by tissue processing and embedding in paraffin and the other portion fixed in 3% glutaraldehyde, followed by processing into epoxy resin. The paraffin embedded tissues were cut at 5-µm thin sections and stained with a battery of histochemical and immunohistochemical stains (including immunostaining for neurofilaments with SMI31 antibody (1:5000; Sternberger, shown in Fig. 3) and the resin-embedded tissues were cut into 1-µm semi-thin sections and stained with methylene blue azure-basic fuchsin. All slides were viewed under light microscope. Histology images shown in the Fig. 3 were digitized on a LEICA SCN400F scanner at ×40 magnification and 65% image compression setting during export. For the preparation of light microscopy images, image captures were taken in LEICA SlidePath (LEICA Milton Keynes, UK). Publication figures were assembled in Adobe® Photoshop.
For the analysis of myelinated fibre density in sural nerve biopsies, four randomly selected fascicles from each case were assessed. Both large and small myelinated fibres were counted.