Specimen Collection for Translational Studies in Hidradenitis Suppurativa

Abstract

Hidradenitis suppurativa (HS) is a chronic inflammatory disorder characterized by painful nodules, sinus tracts, and scars occurring predominantly in intertriginous regions. The prevalence of HS is currently 0.053-4%, with a predominance in African-American women and has been linked to low socioeconomic status. The majority of the reported literature is retrospective, population based, epidemiologic studies. In this regard, there is a need to establish a repository of biospecimens, which represent appropriate gender and racial demographics amongst HS patients. These efforts will diminish knowledge gaps in understanding the disease pathophysiology. Hence, we sought to outline a step-by-step protocol detailing how we established our HS biobank to facilitate the formation of other HS tissue banks. Equipping researchers with carefully detailed processes for collection of HS specimens would accelerate the accumulation of well-organized human biological material. Over time, the scientific community will have access to a broad range of HS tissue biospecimens, ultimately leading to more rigorous basic and translational research. Moreover, an improved understanding of the pathophysiology is necessary for the discovery of novel therapies for this debilitating disease. We aim to provide high impact translational research methodology for cutaneous biology research and foster multidisciplinary collaboration and advancement of our understanding of cutaneous diseases.

Figure 1

Overview of the personalized approach taken to biobank HS samples and the goals of the HS Consortium. Figure produced with Piktochart.

Figure 2

Analysis techniques possible for collected biospecimens. Checks denote whether each analysis technique can be performed with a collected biospecimen.

Figure 3

Examples of analysis techniques for collected biospecimens. (a) Hematoxylin & Eosin staining of HS lesional skin from fresh-frozen tissue. (b) Fontana-Masson staining of HS skin from paraffin-embedded tissue. Scale bar: 100 μm. (c) Immunofluorescence of HS skin for keratin 14 (red), MPO-myeloperoxidase (green), and DAPI (blue). (d) HS skin was digested and primary fibroblasts (first panel) and primary epidermal cells- keratinocytes and melanocytes- (second panel) were cultured. Peripheral blood was collected from HS patients and monocytes/macrophages (third panel) and neutrophils (fourth panel) were isolated and cultured. Scale bar: 50 μm. (e) HS skin was digested into single cell suspension and stained for flow cytometry analysis. (f) Protein was extracted from peripheral blood cells and used to perform Western blot. Uncropped Western Blot in Supplemental Fig. S1. (g) RNA was extracted from homogenized healthy control and HS skin and was quantified with a spectrophotometer. The 260/280 absorbance ratio of ~2 represents the purity and quality of the RNA. (h) Heatmap representing the microbiome of healthy control and HS skin developed by next-generation sequencing.

Figure 4

Supplies for operating room, benchwork, and storage. Listed are the supplies necessary for the operating room, benchwork, and storage when working with HS biospecimens as well as their purpose and manufacturers.

Figure 5

(a) A large piece of freshly resected HS tissue on foil (left). The tissue can be further sectioned using a scalpel to enable proper storage and further studies (right). (b) Arrows indicate areas of apparent nodules and lesions on freshly resected HS tissue.

Figure 6

Comparative analysis of paraffin and fresh frozen tissue with images. Positive and/or negative aspects for each consideration when processing tissue as paraffin or fresh frozen are listed. Images of possible analysis techniques are shown. (+) denotes positive aspect; (−) denotes negative aspect.