Image analysis quantification of peptide‐immunoreactive nerves in the skin of patients with Raynaud's phenomenon and systemic sclerosis

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Abstract
Image analysis quantification was used to assess the results of immunocytochemistry for a neuronal marker and neuropeptides in digital skin biopsies from Raynaud's phenomenon (RP) and systemic sclerosis (SS) patients, to verify the possibility of a selective quantitative abnormality of immunoreactive nerves. The field area of specific immunostaining and nerve counts were evaluated on coded specimens, and the data compared by statistical analysis. Nerves immunoreactive for protein gene product 9.5 (PGP), a marker for neuronal elements, were decreased significantly in epidermal and subepidermal layers of digital skin in RP patients (P < 0.0001). This change was paralleled by a decrease of calcitonin gene‐related peptide (CGRP) immunoreactive nerves in the epidermis and around capillaries in the dermal papillae (P=0.005). In the skin of RP patients, these changes were readily demonstrated by image analysis, although they were not always apparent on visual screening. In digital skin of SS patients, there was a generalized and very significant decrease of PGP, CGRP, and VIP immunoreactivities in all areas (P<0.0001). These results demonstrate that neuropeptide‐containing nerves are involved in the digital pathology of RP and SS, and that image analysis quantification is an accurate and sensitive method for assessing morphological changes in pathological samples.