Protocols and standards used f for histopathology processing, imaging and single-cell sequencing. 1. Histopathology In order to identify the progress of Crohn’s Disease in the patient samples, we are utilising a number of histopathological techniques: Haematoxylin and Eosin (H&E) staining – a gold standard technique used by pathologists to identify any pathological changes to the tissue1,2 . Immunohistochemistry (IHC) – through use of antibodies against specific markers present on the surface of the cells, we can identify the localisation of cells of interest in Crohn’s Disease ileum and colon samples3,4. Picro-Sirius Red staining (PSR) – a technique that allows visualisation of collagen within tissue – crucial in understanding the changes that occur in the late stages of Crohn’s Disease5,6. Examples of Stains: Image Examples of stained Healthy Ileum Image Examples of stained Crohn’s Diseased Ileum As shown in the images, there are substantial pathological changes in the ileum tissue – loss of mucosa, extensive formation of scar tissue (shown by red stain of collagen in PSR) and localisation of immune T-cells as shown by IHC (CD3) staining. 2.Image analysis In order to analyse the stain images, various image-processing software was utilised – specifically, QuPath and ImageJ. QuPath, developed by Dr Peter Bankhead, is an open-source software for bioimage analysis. It is often used for digital pathology application because it offers a powerful set of tools for working with whole slide images7. This powerful software provides a multitude of functions, such as pixel classifiers that can be trained to identify specific cells or tissue (in our case collagen, see below) or detecting and quantifying IHC DAB-positive cells. Image Detection of collagen in the PSR image with QuPath. In parallel, ImageJ (or Fiji), another open-source software was used to analyse microscopy images8. Both pieces of software were used to generate the images shown on this webpage. 3. Single cell RNA sequencing In order to further understand the processes and changes that occur in the late stages of Crohn’s Disease, we are aiming to utilise single cell RNA sequencing with use of 10X Chromium sequencer. References Titford, M. (2005). "The long history of hematoxylin". Biotechnic & Histochemistry. 80 (2): 73–80. DOI:10.1080/10520290500138372. PMID 16195172 Smith C (2006). "Our debt to the logwood tree: the history of hematoxylin". MLO Med Lab Obs. 38 (5): 18, 20–2. PMID 16761865 Ramos-Vara, JA; Miller MA (2014). "When tissue antigens and antibodies get along: revisiting the technical aspects of immunohistochemistry--the red, brown, and blue technique". Veterinary Pathology. 51 (1): 42–87. DOI:10.1177/0300985813505879. PMID 24129895 Ramos-Vara, J. A. (2005). "Technical Aspects of Immunohistochemistry". Veterinary Pathology. 42 (4): 405–426. DOI:10.1354/vp.42-4-405. PMID 16006601 Lattouf R, Younes R, Lutomski D, Naaman N, Godeau G, Senni K, Changotade S. Picrosirius red staining: a useful tool to appraise collagen networks in normal and pathological tissues. J Histochem Cytochem. 2014 Oct;62(10):751-8. DOI: 10.1369/0022155414545787. Epub 2014 Jul 14. PMID: 25023614. RW Dapson, C Fagan, JA Kiernan & TW Wickersham (2011) Certification procedures for sirius red F3B (CI 35780, Direct red 80), Biotechnic & Histochemistry, 86:3, 133-139, DOI: 10.3109/10520295.2011.570277 Bankhead, P. et al. (2017). QuPath: Open source software for digital pathology image analysis. Scientific Reports. DOI:10.1038/s41598-017-17204-5 Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., … Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nature Methods, 9(7), 676–682. DOI:10.1038/nmeth.2019 This article was published on 2024-08-27