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Patients with prurigo nodularis (PN) present with pruritus which may lead to sleep disturbances and systemic comorbidities. The objective of our study was to determine the risk of sleep disorders in PN and its association with systemic inflammation and adverse cardiovascular outcomes. We conducted a retrospective population-level cohort using a global health records database to analyze the development of sleep disorders, C-reactive protein (CRP) levels, and risk of cardiovascular disease and mortality in PN compared to controls. PN patients had increased risk of general sleep disorders (RR 1.47, 95%CI 1.42-1.52, obstructive sleep apnea (1.61, 1.54-1.69), insomnia (1.37, 1.29-1.45), hypersomnia (1.47, 1.32-1.64), and restless legs syndrome (1.45, 1.28-1.65). Additionally, PN with sleep disorders had higher CRP levels than PN without sleep disorders (16.2 mg/L vs. 10.6 mg/L). PN patients with sleep disorders were more likely to develop cardiovascular comorbidities, including type 2 diabetes mellitus (RR 2.45, 95%CI 2.25-2.66), metabolic syndrome (4.16, 3.31-5.22), myocardial infarction (2.87, 2.52-3.22), venous thromboembolism (2.93, 2.55-3.39), and greater mortality (HR 1.47, 95%CI 1.34-1.62) compared to PN patients without sleep disorders. Our findings suggest that PN patients are at greater risk of developing various sleep disorders that are associated with adverse cardiovascular outcomes., Competing Interests: Declarations. IRB status: Waived. Patient consent: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Microenvironment evaluation of intact tissue for analysis of cell infiltration and spatial organization are essential in understanding the complexity of disease processes. The principle techniques used in the past include immunohistochemistry (IHC) and immunofluorescence (IF) which enable visualization of cells as a snapshot in time using between 1 and 4 markers. Both techniques have shortcomings including difficulty staining poorly antigenic targets and limitations related to cross-species reactivity. IHC is reliable and reproducible, but the nature of the chemistry and reliance on the visible light spectrum allows for only a few markers to be used and makes co-localization challenging. Use of IF broadens potential markers but typically relies on frozen tissue due to the extensive tissue autofluorescence following formalin fixation. Flow cytometry, a technique that enables simultaneous labeling of multiple epitopes, abrogates many of the deficiencies of IF and IHC, however, the need to examine cells as a single cell suspension loses the spatial context of cells discarding important biologic relationships. Multiplex fluorescent immunohistochemistry (mfIHC) bridges these technologies allowing for multi-epitope cellular phenotyping in formalin fixed paraffin embedded (FFPE) tissue while preserving the overall microenvironment architecture and spatial relationship of cells within intact undisrupted tissue. High fluorescent intensity fluorophores that covalently bond to the tissue epitope enables multiple applications of primary antibodies without worry of species specific cross-reactivity by secondary antibodies. Although this technology has been proven to produce reliable and accurate images for the study of disease, the process of creating a useful mfIHC staining strategy can be time consuming and exacting due to extensive optimization and design. In order to make robust images that represent accurate cellular interactions in-situ and to mitigate the optimization period for manual analysis, presented here are methods for slide preparation, optimizing antibodies, multiplex design as well as errors commonly encountered during the staining process.