| Summary: | Immunohistochemical quantification of inflammatory cells in skin biopsies is a valuable tool for diagnosing skin diseases and assessing treatment response. The quantification of individual cells in biopsies is time-consuming, tedious, and difficult. In this study, we presented and compared two methods for the quantification of CD8<sup>+</sup> T cells in skin biopsies from patients with psoriasis using both commercial software (Adobe Photoshop) and open-source software (Qupath). In addition, we provided a detailed, step-by-step description of both methods. The methods are scalable by replacing the CD8 antibody with other antibodies to target different cells. Moreover, we investigated the correlation between quantifying CD8<sup>+</sup> cells normalized to area or epidermal length and cell classifications, compared cell classifications in QuPath with threshold classifications in Photoshop, and analyzed the impact of data normalization to epidermal length or area on inflammatory cell densities in skin biopsies from patients with psoriasis. We found a satisfactory correlation between normalizing data to epidermal length and area for psoriasis skin. However, when non-lesional and lesional skin samples were compared, a significant underestimation of inflammatory cell density was found when data were normalized to area instead of epidermal length. Finally, Bland–Altman plots comparing Qupath and Photoshop to quantify inflammatory cell density demonstrated a good agreement between the two methods.
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