Mini‐zone cyanoacrylate skin surface stripping: a new method for non‐invasive sampling of scalp material

Clinical studies in the field of hair research largely rely on clinical read-outs such as hair density, hair shaft thickness or hair growth activity. Efficacy assessments require study durations of several months. Due to the complexity of hair growth regulation, preclinical models are limited. Thus, the inclusion of molecular markers could tremendously help our understanding of hair pathologies and treatment efficacies. Yet, besides scalp skin biopsies and hair plucking, only few sampling methods are established for scalp skin, and there is a need for standardized methods for non-invasive sampling, which yield enough material and can be applied on multiple different investigational sites, for example comparison of affected and unaffected or treated versus untreated skin. Non-invasive sampling methods for hair research face the challenge that sufficient material needs to be collected from small skin surfaces and that such material should be representative for hair follicle processes. On other body sites, tape stripping methods can be conveniently used to collect superficial inflammatory cytokines 1-4, but adhesive tapes only remove superficial skin surface material and corneocytes. In contrast, cyanoacrylate glue flows into hair follicle openings 5. We previously used cyanoacrylate glue to quantify the infundibular volume or topically applied compounds in individual terminal hair openings (cyanoacrylate skin surface stripping, CSSS) 6. Here, we present a standard operating procedure (SOP), which enables investigators to collect material from the scalp skin surface and terminal hair follicle openings. This protocol combines features of skin surface stripping and cyanoacrylate stripping in a ‘miniaturized’ set-up. Because it can be applied on the same investigational sites also used for phototrichogram analyses, it is easily integrated in clinical trials protocols. We call this mini-zone CSSS. Extraction and quantification of proteins from cyanoacrylate glue have not yet been reported. Major challenges are efficient recovery of the protein from CSSS as well as interference of glue material with many colorimetric assays. We show a new approach for the extraction and quantification of protein from CSSS. Protein harvesting using the newly developed mini-zone CSSS sampling method was performed on nine human volunteers (total number of 20 scalp sites) following a SOP illustrated in Fig. 1 (for details on the steps of extraction and data analysis, please refer to Data S1). Institutional review board (Charité-Universitaetsmedizin Berlin) approval and written informed consent were obtained. The study was performed in adherence with the Declaration of Helsinki Principles. Briefly, a plastic template with a 1.9-cm-diameter punch hole was placed on the test area. Using an electrical shaver, hair shafts were cut down to a length of 0.4 mm followed by wet shaving down to scalp level. The mini-zone CSSS template (Fig. 1a) is a tailored silicone template which allows for safe application of liquid glue in a central area of 1.5 cm diameter and subsequent tape application in accordance with standard CSSS protocols. One drop of cyanoacrylate glue (equivalent to 20 mg) was applied in the central area of the shaved investigational site, immediately followed by the CSSS patch, hardening and subsequent removal of the glue resin. After an incubation of 20 min, the silicone/tape patch was quickly removed and stored at −80°C. The attached proteins were eluted by incubation in PBS containing 0.005% Tween-20 for 3 h followed by sonication, quantified using an amido black protein assay (Prota Quant, Serva Electrophoresis GmbH (Heidelberg, Germany)) and subjected to protein array analysis. The mean total protein amount of 11.002 μg/ml in the extracted scalp material was 6 times higher than extracts from Sebutapes taken from arm skin (Sebutape area: 5.44 cm2, mini-zone CSSS area: 1.76 cm2). Protein arrays for cytokine (Human Cytokine Array Kit Panel A, R&D Systems (Minneapolis, MN, US)) and chemokine (Human Chemokine Array Kit, R&D Systems) were used. Microscopic analysis and our previous experimental work on CSSS 5 confirm the presence of casts from hair follicle infundibulum. For cytokine and chemokine detection, samples were normalized to the total protein amount (Fig. 2). Protein profile analyses revealed a wide range of cytokines and chemokines including IL-1α, IL-1RA, IL-13 and MIF (macrophage migration inhibitory factor), CX3CL1 (Fractalkine), chemerin, 6Ckine, MCP-1, CXCL10 (IP-10), MIP-3a, MIP-3b, CCL22 (MDC), TARC and PARC. Semi-quantitative analyses of the spot densities showed meaningful differences among the donors, suggesting that this methodology is reproducible and could be used to compare different scalp samples. The material was also sufficient to perform protein profiles as well as quantitative single-molecule ELISA for 2-3 markers in duplicates. Mean IL-1alpha and IL-1RA (human IL-1alpha, IL-1ra, R&D Systems) for 18 samples were 3.25 ± 0.2 pg/μg total protein and 121.99 ± 1.8 pg/μg total protein, respectively. The presence of IL-1α and IL-1RA is in accordance with published data from Perkins M., who recovered scalp material using Sebutape 1, 7. This result indicates the potential of this methodology for the identification of markers related to scalp inflammation and immune activation 8, 9. In previous studies, we introduced mini-zones for intra-individual comparisons of hair growth in human volunteers. The herein presented mini-zone CSSS sampling method can be conducted on such mini-zones, integrated in clinical protocols and reproducibly yields protein material sufficient not only for array analyses but also for conventional quantitative ELISA. Because the method is non-invasive and painless, it can be performed on multiple scalp sites at a time, also repetitively at the same scalp sites, for example once per month. It could, thus, become helpful for the investigation of cicatricial alopecia and for investigations in cosmetically sensitive areas where volunteers would be disturbed by plucking of large number of hairs or by scarring due to biopsies. Future applications include exploratory studies on disease pathophysiology as well as addition of selected early and longitudinal read-outs in clinical trial settings. AV designed the project, EBP conducted experiments, wrote the manuscript and analysed data, SH, ND, SF conducted experiments and UBP contributed to data analysis. This work is an investigator-initiated research project designed and conducted by the authors. We thank the Hair and Skin Research Institute GmbH and Johnson & Johnson Consumer & Personal Products Worldwide for financial support. Data S1. Material and methods. Data S2. Supplementary References. Table S1. Mean pixel density and standard deviation of positive chemokines, measured using the Protein Profiler, Human Chemokine Array Kit from R&D Systems. N is the number of positive probes in the array. Table S2. Mean pixel density and standard deviation of positive cytokines, measured using the Protein Profiler, Human Cytokine Array Kit Panel A Kit from R&D Systems. N is the number of positive probes in the array. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.