Quantification of Immunohistochemically Stained Cells in Skin Biopsies

Area of Science:

• Dermatology
• Immunohistochemistry
• Digital Pathology

Background:

• Immunohistochemical quantification of inflammatory cells in skin biopsies aids diagnosis and treatment assessment.
• Manual cell counting in biopsies is laborious and prone to error.
• Automated quantification methods are needed to improve efficiency and accuracy.

Purpose of the Study:

• To present and compare two methods for quantifying CD8+ T cells in psoriasis skin biopsies using Adobe Photoshop and Qupath software.
• To investigate the impact of normalization (area vs. epidermal length) on inflammatory cell density.
• To assess the agreement between commercial and open-source software for cell quantification.

Main Methods:

• Detailed step-by-step protocols for CD8+ T cell quantification using Photoshop and Qupath.
• Comparison of cell classification methods (threshold vs. QuPath classification).
• Analysis of normalization strategies (area vs. epidermal length) and Bland-Altman plots for method agreement.

Main Results:

• Satisfactory correlation found between normalizing CD8+ T cell counts to epidermal length and area in psoriasis skin.
• Significant underestimation of inflammatory cell density occurred when normalizing to area versus epidermal length in lesional vs. non-lesional skin.
• Bland-Altman analysis showed good agreement between Qupath and Photoshop for quantifying inflammatory cell density.

Conclusions:

• Both Photoshop and Qupath offer reliable methods for quantifying CD8+ T cells in skin biopsies.
• Normalization to epidermal length is recommended over area for accurate inflammatory cell density assessment, especially when comparing lesional and non-lesional skin.
• The presented methods are scalable for quantifying other cell types by substituting antibodies.