Microbiopsy engineered for minimally invasive and suture-free sub-millimetre skin sampling

Abstract

We describe the development of a sub-millimetre skin punch biopsy device for minimally invasive and suture-free skin sampling for molecular diagnosis and research. Conventional skin punch biopsies range from 2-4 mm in diameter. Local anaesthesia is required and sutures are usually used to close the wound. Our microbiopsy is 0.50 mm wide and 0.20 mm thick. The microbiopsy device is fabricated from three stacked medical grade stainless steel plates tapered to a point and contains a chamber within the centre plate to collect the skin sample. We observed that the application of this device resulted in a 0.21 ± 0.04 mm wide puncture site in volunteer skin using reflectance confocal microscopy. Histological sections from microbiopsied skin revealed 0.22 ± 0.12 mm wide and 0.26 ± 0.09 mm deep puncture sites. Longitudinal observation in microbiopsied volunteers showed that the wound closed within 1 day and was not visible after 7 days. Reflectance confocal microscope images from these same sites showed the formation of a tiny crust that resolved by 3 weeks and was completely undetectable by the naked eye. The design parameters of the device were optimised for molecular analysis using sampled DNA mass as the primary end point in volunteer studies. Finally, total RNA was characterized. The optimised device extracted 5.9 ± 3.4 ng DNA and 9.0 ± 10.1 ng RNA. We foresee that minimally invasive molecular sampling will play an increasingly significant role in diagnostic dermatology and skin research.

Conflict of interest statement

Competing interests: No competing interests were disclosed.

Figures

Figure 1.. Size comparison of needle, biopsy devices and biopsy comparisons.

A conventional biopsy punch is shown on the left, an 18 gauge syringe needle in the centre and the inner chamber of our microbiopsy device on the right Panel (a) our microbiopsy device chamber is 0.15 mm in width with an outer width of 0.25 mm. The top row of Panel (b) contains a conventional 3 mm biopsy site and tissue, whereas the bottom panels show microbiopsied skin and tissue.

Figure 2.. Microbiopsy channel width and velocity optimisation.

Channel width and velocity were varied to optimise the microbiopsy device configuration. Total DNA was used as a surrogate for sample size. Panel (a) shows the DNA extracted from device with varying channel width and that the maximum amount of DNA was collected with 0.15 mm channel width. Panel (b) displays high resolution scanning electron microscopic images showing different channel widths of the microbiopsy device. Panel (c) shows the level of pain volunteers reported when different channel width microbiopsies were used. Panel (d) shows the varying velocity applied and that the maximum amount of DNA was collected when the device was applied at 16.6 m/s. Panel (e) shows the level of pain volunteers reported when application velocity was varied.

Figure 3.. Microbiopsy roughness amplitude optimisation.

Panel (a) shows that microbiopsy devices with higher roughness amplitude channels are capable of collecting more DNA. Panel (b) contains high resolution scanning electron microscopic images showing different channel widths and roughness of the microbiopsy device.

Figure 4.. Site of microbiopsy and microbiopsy content.

Panels (a) and (b) are reflectance confocal microscopy mosaics of a microbiopsy site, see the hair follicles featured in the centre and on the right hand side of the images for size comparison (bar indicates 0.5 mm ina andb). Panel (c) shows a 63x magnification, 3D rendering of the microbiopsy tissue with a nuclear counter stain (orange) derived from a confocal microscopy z-stack of the sample within the microbiopsy device. The stratum corneum (SC), viable epidermis (VE), dermal-epidermal junction (DEJ) and superficial dermis (DER) are labeled. This microbiopsy contained an estimated 1634 nuclei. Haematoxylin and eosin stained section of human skin after microbiopsy application shows a 0.10 mm wide and 0.25 mm deep puncture Panel (d). * indicates the site of microbiopsy application.

Figure 5.. Molecular characterization of conventional shave biopsies and microbiopsies.

The left panel shows the Bioanalyzer readout from amplified DNA from a conventional actinic keratosis (AK) shave biopsy next to the microbiopsy DNA sample obtained from the same AK lesion. The middle panel shows the Bioanalyzer readout from RNA isolated from a conventional AK shave biopsy and a microbiopsy that was obtained from the same AK lesion. The right panel compares the RNA quality of a shave biopsy and normal skin microbiopsy that were subjected to total transcriptome amplification to generate cDNA. The cDNA was then used as a template for a PCR reaction containing actin specific primers with an expected product at 800 bp.

Figure 6.. Wound healing kinetics of microbiopsy site.

The left column shows dermoscopic images of the microbiopsy site over time. The middle and right column are mosaics and at 30x magnification reflectance confocal microscopy (RCM) images, respectively, of the microbiopsy site.