Advancement through epidermis using tape stripping technique and Reflectance Confocal Microscopy

The tape stripping technique is increasingly used in research regarding skin barrier function. However, number of tape strips varies between studies, and literature considering advancement into stratum corneum/epidermis in relation to number of tape strips is scarce. The aim of this pilot study was to assess the advancement through epidermis using tape stripping technique in healthy volunteers. A total of ten healthy volunteers were included. From all volunteers 0, 5, 15 and 35 consecutive tape strips (D-squame) were taken from four adjacent skin areas on the middle volar forearm, followed by Reflectance Confocal Microscopy (RCM) of the four areas to assess epidermal thickness. Squame Scan was used to determine amount of protein removed. Stratum corneum was completely removed in all volunteers after 35 tape strips. Advancement into epidermis was predominantly achieved by the first 15 tape strips, removing 25% of the total epidermis, whereas 35 tape strips removed 33% of epidermis. Protein removal per tape decreased with increasing depth. Information on advancement into the epidermis according to number of tape strips taken, is a significant step forward. The possibility to obtain samples from different layers of epidermis may lead to an improved understanding of skin barrier properties.

Stratum corneum was completely removed after 35 tape strips in all volunteers as evaluated visually and by RCM. After five tape strips stratum corneum was removed in none of the volunteers, after 15 tape strips stratum corneum was removed in only one volunteer.

Discussion
The tape stripping technique has recently attracted much attention, since the method is a minimal invasive technique useful for sampling of epidermis and has a significant advantage to skin biopsies with respect to discomfort for patients and volunteers. Although increasingly used, the exact advancement into epidermis following tape stripping has remained unknown. In the present study this was explored by RCM, which clearly showed that the stratum corneum had been completely removed after 35 strips in all volunteers, with a removal of one third of the total epidermis. While 11% of the epidermis was removed after the first five tape strips, and 25% was removed after 15 tape strips, the additional 20 tapes did not advance to the same degree into the epidermis. Increasing cohesion between cells when advancing into the epidermis is likely to explain this as the first five tape strips caused a decrease in the epidermal thickness by 6.4 µm, whereas the last 20 tape strips decreased the epidermal thickness only by 4.4 µm. Increased cohesion in the deeper layers of epidermis has been reported in previous studies 14,15 . Although gender ratio in this study was skewed with 70% women and 30% men, it is not expected to influence the results, as previous studies have shown that gender does not influence tape stripping with respect to protein data 16 . A correlation was found between protein removal and decrease in epidermal thickness. Interestingly, it seems as there is a larger variation in protein removal in the deeper layers (T15 and T35) compared to the upper layers (T5) (Fig. 3). This likely reflects individual differences in strength and structure of the epidermis, as some participants seem to have a skin that very easily strips off when visually evaluating the tapes, as compared to others. In addition, the Squame Scan only scans the middle area of the tape corresponding to 50% of the total tape, and accordingly, uneven attachment of the skin to the tape, might cause a poor correlation between protein determination and decrease in epidermal thickness.
Until now an important bias related to tape stripping has been the uncertainty of how deep into the epidermis the method reaches, and methods like visual assessment, measurement of TEWL and protein content have not proved sufficient for solving this problem. Previously the appearance of red glistening skin and/or significant increase in TEWL have been interpreted as the removal of stratum corneum. Using the RCM to follow the gradual tape stripping of stratum corneum, we here present new data illustrating step by step how far down into the epidermis the method reaches. These data are new and necessary basic data for future use of tape stripping in the investigation of the skin barrier.
An important limitation to the tape stripping method is that skin furrows may complicate an exact differentiation of different layers in the epidermis 17 . Furthermore, the method is aimed predominantly at the skin barrier (stratum corneum), as reaching deeper levels might be challenging.

Conclusion
Tape stripping is useful for skin barrier research and has the advantage over skin biopsies that it allows for collection of stratum corneum without scars and discomfort for the participant/patient. Results from the present study give a clear indication of advancement into epidermis according to number of tape strips, and open for the possibility to evaluate differences in skin barrier properties in different parts of the epidermis.

Methods
Participants. Healthy volunteers were recruited from the Department of Dermatology, Bispebjerg Hospital, Copenhagen. Topical application of emollients or other topical treatments was not allowed on the day of skin sampling.
Tape stripping. Each participant had 0, 5, 15 and 35 consecutive tape strips (D-squame 3.8 cm 2 ) (CuDerm) taken from four adjacent skin areas on the middle volar forearm (non-lesional skin). Each tape was pressed www.nature.com/scientificreports www.nature.com/scientificreports/ against the skin for 10 sec with standardized pressure (225 gr./cm 2 ), using the D-squame pressurizer (CuDerm), before stripped from the skin. Localization for tape stripping was randomized from a planned system to four adjacent skin areas 4, 8, 12, 16 cm from the wrist, respectively.

Reflectance confocal microscopy (RCM).
A commercially available reflectance confocal microscope (Vivascope Multilaser 1500 ® , Caliber ID Inc., Rochester, NY, USA) was applied in 658 nm mode. The RCM provides real-time horizontal images of the skin to a depth of 200-250 µm, which includes the entire epidermis and upper papillary dermis. In each test area, i.e. 0, 5, 15 and 35 tape strips respectively, 3 individual z-stacks stacks consisting of horizontal 500 × 500 µm optical sections were acquired at every 1.5 µm from skin surface to a depth of 82 µm, resulting in 53 images per z-stack.
RCM z-stacks were used to identify stratum corneum, stratum granulosum, stratum spinosum and the basement membrane; the latter with visible papillae in it. To assess epidermal thickness after 0, 5, 15, and 35 tape strips, the depth from the surface of the skin to the first visible papillae in the basement membrane was assessed in RCM images. To take variation of the papillae length into consideration, we decided that three papillae should be visible in the z-stack before measuring the depth. Measurements were performed in all three z-stacks from each test area and mean depth from skin surface to basement membrane represented epidermal thickness.
Skin barrier function. Skin barrier function was assessed by measurements of TEWL, at the middle volar forearm before tape stripping and after 35 tape strips (DermaLab, Cortex Technology, Denmark). Participants adjusted to room temperature before TEWL measurements. Skin pH was measured at the middle volar forearm