Dec 07, 2023

Public workspaceCD3 Cell Density in Substantia Nigra and Cerebral Peduncle Image Analysis

DOI
dx.doi.org/10.17504/protocols.io.dm6gp3j9pvzp/v1
  • 1Department of Clinical and Movement Neurosciences, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20 815, USA;
  • 3Queen Square Brain Bank for Neurological Disorders (QSBB), 1 Wakefield Street, London WC1N 1PJ, UK
Hemanth Ramesh Nelvagal
Open access
DOI: dx.doi.org/10.17504/protocols.io.dm6gp3j9pvzp/v1
Protocol Citation: Hemanth Ramesh Nelvagal, Toby J Curless, Zane Jaunmuktane 2023. CD3 Cell Density in Substantia Nigra and Cerebral Peduncle Image Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp3j9pvzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 07, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 86037
Keywords: ASAPCRN, Human Brain, Substantia Nigra, Cerebral Peduncle, CD3, Parkinson’s disease, QuPath, NZConnect, Image Analysis, Pathology
Funders Acknowledgement:
The Michael J. Fox Foundation for Parkinson’s Research (MJFF) and the Aligning Science Across Parkinson’s (ASAP) Initiative
Grant ID: ASAP-000478
Abstract
QuPath is a bioimage analysis software designed for digital pathology and whole slide image analysis. This protocol describes how to measure CD3 density in the substantia nigra and cerebral peduncle using haematoxylin and DAB-stained brain sections.
Materials
  • CD3 IHC-stained sections
  • NZConnect (Hamamatsu)
  • StarDist
  • QuPath
Annotation
Annotation
Manually annotate the substantia nigra and cerebral peduncle on NZConnect (Hamamatsu), a web-based whole-slide image (WSI) viewer.
Download the annotations using a Python script then import into QuPath [1] using a Groovy script.

QuPath Deconvolution and CD3 Density Measurement
QuPath Deconvolution and CD3 Density Measurement
In QuPath, set the colour deconvolution to facilitate the detection of haematoxylin and DAB staining on CD3 IHC-stained sections.
Segment all cell nuclei using StarDist [2] via the QuPath StarDist extension [3];followy with an object classifier to classify CD3-positive cells.
Calculate CD3-positive cell density by the number of CD3-positive cells divided by the area of the region of interest (CD3-positive cells per mm^2).
Protocol references
[1] Bankhead, P., Loughrey, M.B., Fernández, J.A. et al. QuPath: Open source software for digital pathology image analysis. Sci Rep 7, 16878 (2017). https://doi.org/10.1038/s41598-017-17204-5

[2] Schmidt, U., Weigert, M., Broaddus, C., & Myers, G. (2018, September). Cell detection with star-convex polygons. In International Conference on Medical Image Computing and Computer-Assisted Intervention (pp. 265-273). Springer, Cham. https://arxiv.org/abs/1806.03535
[3] StarDist extension for QuPath
https://github.com/qupath/qupath-extension-stardist