Microcore Skin Biopsies: A Novel, Micro‐Invasive Skin Biopsy Technique Yielding Full‐Thickness Histology

ABSTRACT

Background

Punch biopsies are considered the primary technique to obtain full‐thickness skin specimens for dermatologic diagnoses. However, limitations include scarring, sampling error, poor margin identification, and post‐sampling deformations.

Objectives

To introduce a novel micro‐biopsy technique utilizing a modified hypodermic needle to achieve full‐thickness skin micro‐columns and a streamlined post‐sampling process for histological analysis.

Methods

Ex‐vivo skin biopsies were taken using a modified hypodermic needle and compared to 3 mm punch biopsies. They were fixed and processed inside the needle, resulting in a firm, columnar sample, which was then sectioned, stained, and imaged. Dimensions and horizontal shrinkage were also calculated.

Results

The technique consistently yielded uniform, columnar‐shaped histological samples. These sections were of sufficient quality to perform staining with a significantly smaller horizontal shrinkage rate compared to standard 3 mm punch biopsies.

Conclusions

We demonstrate a novel technique for a skin micro‐biopsy that allows for fixation and processing of the tissue inside the sampling instrument, yielding small, full‐thickness columnar skin histological samples that can be used for dermatological diagnosis with minimal post‐sampling deformation.

Plain Language Summary

Punch biopsies are considered the primary technique to obtain full‐thickness skin specimens in order to diagnose skin conditions. However, scarring associated with skin biopsies and the deformations that happen to samples during processing pose persistent challenges. To minimize invasiveness, sub‐millimeter skin punch biopsies have been introduced, but there has been no standardized, widely adoptable technique to utilize sub‐millimeter skin punch biopsies to make diagnoses. Here, we introduce a novel skin biopsy needle that obtains skin samples that are less than 0.5 mm in diameter, which is below the scarring threshold, as well as a processing technique to easily fix, stain, and image the samples to make skin diagnoses.

1. Introduction

Punch biopsies are considered the primary technique to obtain skin specimens that are full‐thickness and that can be imaged in order to make dermatologic diagnoses [1]. Disposable or sterilizable punch biopsy instruments can be obtained of various sizes; however, punch biopsies of 2–4 mm are the commonly used sizes [2, 3]. This allows for visualization and accurate diagnosis of a multitude of skin pathologies.

To minimize scarring and other complications, careful suturing, appropriate dressing, and close observation are required, especially in certain clinical situations such as skin‐of‐color patients, exposed skin regions, or inflammatory skin conditions. However, despite taking extreme caution, complete healing without any noticeable scarring is difficult to achieve [4]. Noticeable scarring reduces patient satisfaction, as they are generally regarded as unsightly [5]. Additionally, punch biopsies commonly undergo post‐sampling deformations including crush artifact and horizontal shrinkage, which may be exacerbated by physical stresses during and after tissue sampling [6].

To minimize the invasiveness, there have been several experimental attempts to implement the skin micro‐biopsy. There was a laser‐fabricated micro‐biopsy apparatus, first introduced in 2013 by Lin et al. [7], which was able to obtain small skin tissue wedges with ∼400 skin cells to a depth of ∼250 µm [7, 8, 9, 10]. However, the height of the samples from this technique could not reach adequate depth for broad diagnostic application impeding a wide clinical use. Recently, Kislevitz et al. showed that a sub‐millimeter flat‐top micro‐punch can be used to obtain columnar skin samples through deeper depth [11]. Nonetheless, dermatologists have not been able to reach widespread adoption of sub‐millimeter columnar skin biopsy in clinical settings. This is because there is still an open question about how to execute intact histological sample preparation after sample acquisition as the columnar samples are more prone to being lost or deformed as they become smaller and thinner.

On the other hand, the recently introduced minimally invasive micro‐coring system for skin tightening and rejuvenation [12, 13] utilizes hypodermic needles modified to have two‐prong tips by cutting and shaping the needle on two sides. The two‐prong tip of this needle allows this system to core full thickness sub‐millimeter skin columns without a rotating motion, reducing sheer stress to the sample in contrast to conventional flat‐topped circular trephines. Although it can be anticipated that cutting the hypodermic needle to have a two‐pronged tip will help this tool to harvest fragile, slender skin micro‐cores more reliably, there have been no reports of its use as a micro‐incisional biopsy tool, suggesting that a stable and widely useable post‐sampling process has not yet been established.

Therefore, this study aims to introduce a novel micro‐biopsy technique utilizing a hypodermic needle modified to have two distinct features: the two point‐edged prongs and side wall windows, which enable easy sampling of full‐thickness skin micro‐columns and in situ tissue fixation and processing for histological analysis.

2. Materials and Methods

2.1. Production of the Micro‐Coring Needles

For the primary modification, the two‐point prongs were made by carving the tip of 22‐gauge needles according to the protocol described by Tam et al. [14]. Briefly, a 22‐gauge hypodermic needle approximately 12 mm in length was positioned inside a rotary tool and a translation stage was used to advance the needle toward a diamond cutting disc until the cutting disc reached the midline of the needle. The needle was moved away from the cutting disc, rotated 180° and, similarly, advanced until the cutting disc reached the midline of the needle from the opposite direction. The needle was then rotated 45° consecutively on each side to create four additional planes resulting in a sharp point at the tip of each prong. This results in a modified hypodermic needle with a sharp 2‐prong tip that can be driven through the skin vertically.

For the secondary modification, four parallel rows of four holes were drilled on the side wall of the needles (Resonetics, Co., NH, USA). One row of holes aligns with the pointed tips and each hole has a 0.4 mm diameter with a pitch of 0.5 mm and a nominal angle for the tip of 12° (Figure 1a–c). The presence of these holes enables the agents and solutions for tissue fixation and processing to penetrate the needle wall and immerse the skin specimen inside.

FIGURE 1.

Image of the 22G hypodermic needle modified to have a sharp 2‐prong tip and rows of holes in the side wall arranged in parallel (a–c). Image of a conventional 3 mm punch biopsy instrument (d). (Scale bar = 200 µm for a–c and 1 mm for d).

2.2. Ex Vivo Human Skin and Ethical Considerations

This study was conducted in accordance with the ethical standards of the Mass General Brigham Institutional Review Board (IRB). After IRB approval was obtained (IRB No. 2017P000027), human skin specimens were obtained from the excess tissues removed during elective abdominoplasties performed at Massachusetts General Hospital. The human tissue samples would otherwise have been discarded and were fully anonymized before delivery to our laboratory, with no identifying information of the patient retained. Because the tissue came from discarded samples under IRB‐approved exemption specifying no identifiers, no linkage to patient records, and no consent resulting in a violation of privacy, the patients the samples came from could not be tracked or counted.

2.3. Collection of Columnar Skin Specimens

2.3.1. Micro‐Cores

The surface of the skin was gently cleaned with 95% ethanol wipes to remove excess blood and debris on the surface. The skin was pulled taut to create tension and a 22‐gauge micro‐coring needle was positioned perpendicular to the surface of the skin and driven vertically through the skin through the epidermis, dermis, and subcutaneous tissue in a rapid motion. The needle was then slowly removed from the skin. All needles were modified to have the two‐prong tip. Sampling was done with needles both with and without the holes in the side wall for comparison.

2.3.2. Punch Biopsies

Size 3 mm biopsy punches, the most widely chosen apparatus for clinical situations [15], were also taken from the skin using a standard methodology for taking punch biopsies. Briefly, the punch biopsy instrument (Figure 1d) was positioned vertically on the skin and rotated both clockwise and counter‐clockwise alternatively using slight downward pressure and a twirling motion until the punch biopsy instrument penetrated the skin layers [3].

2.4. Post‐Sampling Processes

2.4.1. Micro‐Cores Processed Inside Needles

With the skin samples still inside the micro‐coring needles, the needles were placed into 10% buffered formalin mixed with several drops of eosin and fixed in this solution overnight at room temperature. Then, the entire needle with the samples still inside was placed inside a CellSafe Biopsy Capsule, which was placed into a cassette and processed in a tissue processing machine using the standard operational protocol with the short (approximately 2‐h) processing time setting (Excelsior ES Tissue Processor, Thermo Fisher Scientific, Co., MA, USA). Therefore, the sample was completely infiltrated with paraffin wax. Next, a blunt 28‐gauge needle with its external diameter clearly narrower than the 413 µm internal diameter of the micro‐coring needle [16], was inserted into the 22‐gauge needle to push out the now fixed and processed micro‐core from the needle. The firm micro‐cores were then embedded in paraffin wax (Surgipath Paraplast, Leica Biosystems, Co., CA, USA) and a microtome was used to section the blocks at cut 5 µm. The sections were mounted onto poly‐L‐lysine glass slides (Newcomer Supply, Co., WI, USA). Figure 2 depicts a detailed scheme of the methodology used to harvest, process in paraffin, and section the microcore samples.

FIGURE 2.

Detailed depiction of the skin sample harvesting, processing, and sectioning process.

2.4.2. Micro‐Cores Processed Outside Needles

Immediately after harvesting skin samples using the micro‐coring needle, a blunt 28‐gauge needle was used to remove the samples and place them on filter paper to be collected. The filter paper was then folded and placed inside a cassette and then underwent the same fixation and processing steps as the other samples still inside the needles.

2.4.3. Punch Biopsies

As in a typical biopsy process, the tissue was removed from the punch biopsy instrument and submerged in 10% buffered formalin for tissue fixation overnight at room temperature. Then, the sample was placed into a tissue processing cassette for the tissue processing step. After tissue processing using the same machine as for the micro‐core, the sample was embedded in paraffin wax, and sectioned at 5 µm thick, similar to the microcore samples. The sections were mounted onto poly‐L‐lysine glass slides.

2.5. Staining and Histologic Examination

All chemicals used were purchased from Thermo Fisher Scientific. Both the micro‐cores and the 3 mm punch biopsy specimens were stained with hematoxylin and eosin (H&E) as well as several representative special stains commonly used in dermatology. These stains included the Verhoeff van Gieson elastin stain, the periodic acid‐Schiff stain (PAS), and the Masson's trichrome stain. The immunohistochemical S‐100 stain (Invitrogen) was also performed with a dilution factor of 1:1000. Standard protocols described in the data sheets accompanying the staining kits (Newcomer Supply, Co., WI, USA) used for each of the special stains and immunohistochemistry study were used. The slides were then visualized using a digital slide scanner (NanoZoomer S60; Hamamatsu Photonics Co., Shizuoka, Japan).

2.6. Measurement of Dimensions of Scanned Histological Sections

The length of five micro‐cores and five 3 mm punch biopsies was measured from the scanned histological section and subsequently divided by five to determine the representative depths for taking the five horizontal diameter measurements. The initial measurement was obtained at the superior aspect of the epidermis, followed by four additional measurements, each taken at uniform intervals from the preceding measurement. These measurements were plotted on a graph and compared to the internal diameter of the instrument used to take the biopsies to calculate the horizontal shrinkage that samples undergo after removal from the skin. The lengths that were measured of each biopsy were also used to calculate the mean length of the micro‐core and the 3 mm punch biopsy for comparison.

2.7. Measurement of Wound Sizes

A modified micro‐coring needle as well as a 3 mm punch biopsy instrument were used to take their respective biopsies adjacent to the each other on a skin sample. This created the corresponding wounds, which could then be measured and compared.

2.8. Statistical Analysis

The data analysis was performed using statistical software (GraphPad Prism version 10, GraphPad Software, Co., MA, USA). The mean values were calculated by arithmetic average and shown with the standard deviation and coefficient of variation. Shrinkage measurements from microcore and 3 mm punch samples were paired by matching samples obtained from the same skin specimen. For each pair (n = 5), width was measured at five predefined points and averaged. A paired t‐test was performed using GraphPad Prism, with individual variance and false discovery rate correction (two‐stage step‐up method, Q = 1%).

3. Results

The design of the micro‐coring needle containing a 2‐prong tip and holes in the sidewall enabled fixation and processing of skin samples inside the needle by placing this entire apparatus into the tissue processing cassette (Figure 3a,b). Using this micro‐coring needle, human adult abdominal skin samples were collected, fixed, processed, and removed from the needle with a 28 gauge blunt needle (Figure 3c), yielding cylindrical columns of skin (Figure 3d). It should be noted that, similar to punch biopsy specimens, subcutaneous fat may or may not be present in the grossly extracted microcore sample depending on the plane of shearing during removal.

FIGURE 3.

Image of the modified hypodermic needle with the skin sample inside in the tissue processing cassette (a,b). Image of the final processed skin sample on the guidewire used for removal (c) resulting in firm, columnar micro‐cores of skin (d). (Scale bar = 400 µm for d).

3.1. H&E Staining

The skin samples that were fixed and processed inside the micro‐coring needles readily underwent sectioning and staining with H&E, demonstrating the full‐thickness nature of the skin microcores (Figure 4a–d). These micro‐cores demonstrate a consistent shape (Figure 4a–d), reflecting the appearance of the processed samples when they removed from the micro‐coring needle, just prior to embedding (Figure 3c,d). Histologically, the micro‐cores demonstrate typical features. The epidermis is stratified with a typical basket‐weave pattern to the stratum corneum and a visible stratum granulosum layer (Figure 4e). In the upper dermis, sparse immune cells and superficial vessels can be visualized (Figure 4e). In the mid‐dermis, organized collaged bundles with several fibroblasts and a segment of the eccrine sweat duct can be observed (Figure 4f). In the most inferior aspect of the micro‐core, part of the subcutaneous fat layer can be seen adjacent to the reticular dermis (Figure 4g).

FIGURE 4.

Micro‐cores that underwent hematoxylin and eosin (H&E) staining demonstrate a consistent columnar shape (a–d). At the most superior aspect (e) the typical basket‐weave pattern of the stratum corneum and stratified epidermal layer including the stratum granulosum are observed, as well as rete ridges at the epidermal‐dermal junction and sparse immune cells and superficial vessels. In the middle aspect (f) there are compact horizontally oriented collagen bundles with several fibroblasts and a segment of the eccrine duct. In the lower aspect (g), a part of subcutaneous fat layer is shown at the lower border of the reticular dermis. (Scale bar = 250 𝜇m for a–d and 50 𝜇m for e–g).

The micro‐core samples obtained from the micro‐coring needles without the holes in the side wall demonstrated inferior and less consistent histological results obtained from the samples that came from needles with the holes in the side wall and were significantly more difficult to work with (Figure 5a,b). The micro‐core samples that were removed from the needle prior to fixation and processing were difficult to work with in their fragile state and were easily distorted by the blunt needle during removal (Figure 5c), yielding poor quality histological images (Figure 5d–f).

FIGURE 5.

Hematoxylin and eosin (H&E) staining of micro‐cores that underwent fixation and processing within needles without holes in the sidewall (a,b) with poor tissue quality and poorly discernible features of the epidermis and dermis. Freshly removed micro‐core prior to fixation and processing that is crushed by the blunt needle upon removal (c). H&E staining of micro‐cores fixed and process completely outside of a micro‐coring needle with very poor tissue quality, poorly discernible features of the epidermis and dermis, and breakage (d‐f). (Scale bar = 175 𝜇m for a & b, 200 µm for c, 50 𝜇m for d, and 200 µm for e & f).

3.2. Additional Histological Staining

To demonstrate the versatility of staining that is possible using micro‐cores obtained from our novel harvesting and processing technique, three histological stains were performed. Each of these stains on the microcores exhibits distinct and distinguishable results, which can be used to assess the tissue architecture for diagnostic purposes. The PAS stain highlighted the basement membranes of the epidermis and superficial vessels (Figure 6a). The Masson's trichrome stain revealed dermal collagen fibers in a green hue, differentiating them from other components stained in red (Figure 6b). The Verhoeff van Gieson elastin stain demonstrated elastin fibers in black with collagen stained in a magenta color (Figure 6c).

FIGURE 6.

Micro‐core biopsy sections were stained with periodic acid‐Schiff (PAS), Masson's trichrome, and Verhoeff van Gieson elastin stains (a–c, respectively). PAS staining (a) highlights the basement membranes of the epidermis and superficial vessels. Masson's trichrome staining (b) reveals dermal collagen fibers in a green hue with other components stained in red. Verhoeff van Gieson elastin staining (c) stains elastin fibers in black, while collagen stains in magenta color. (Scale bar = 50 𝜇m).

3.3. Comparison of Wound Size, Histological Width, Horizontal Shrinkage, and Histological Length

The micro‐coring needle used was a standard 22 gauge needle with an internal diameter of 413 µm [16], approximately 14% of that of the 3 mm punch biopsy instrument used. Consequently, the micro‐coring needle made a notably smaller wound (0.13 mm²) compared to the 3 mm punch (7.07 mm²) (Figure 7a), demonstrating the difference in the internal diameters of both instruments (Figure 7b).

FIGURE 7.

Comparison of wound size created by the micro‐coring needle and the 3 mm punch biopsy instrument (a) as well as the internal diameter of both apparatuses (b). (Scale bar = 1 mm).

The micro‐cores demonstrated a 331.1 ± 41.7 µm histological width while the punch biopsy samples showed a 1484.6 ± 159.6 µm histological width (Tables 1 and 2). Interestingly, the microcores had a slightly lower coefficient of variation in histological width compared to the punch biopsies (8.0% vs. 10.75%, respectively).

TABLE 1.

Histologic width of microcore samples.

	Microcore samples
	1	2	3	4	5	Average (𝜇m)	SD† (𝜇m)	CV‡ (%)
Point 1 (𝜇m)	402	317	373	403	399	378.8	36.7	9.7
Point 2 (𝜇m)	325	240	342	316	344	313.4	42.7	13.6
Point 3 (𝜇m)	377	291	323	303	338	326.4	33.6	10.3
Point 4 (𝜇m)	305	300	395	291	346	327.4	43.3	13.2
Point 5 (𝜇m)	301	290	397	302	317	321.4	43.4	13.5
Mean (𝜇m)	329.2	283.6	375.2	318.6	348.8	331.1
SD† (𝜇m)	45.2	28.7	32.7	45.6	30.3		41.7*
CV‡ (%)	13.7	10.1	8.7	14.3	8.7			12.6*

^† Standard deviation; ^‡ Coefficient of variation (SD ÷ Mean × 100); ^*value from overall 25 data.

TABLE 2.

Histologic width of 3 mm punch biopsy samples.

	3 mm punch biopsy samples
	1	2	3	4	5	Average (𝜇m)	SD † (𝜇m)	CV ‡ (%)
Point 1 (𝜇m)	1511	1695	1755	1546	1591	1619.6	102.5270696	6.330394517
Point 2 (𝜇m)	1487	1412	1513	1697	1755	1572.8	146.1307634	9.29112178
Point 3 (𝜇m)	1272	1224	1333	1553	1604	1397.2	170.9055295	12.23200182
Point 4 (𝜇m)	1386	1377	1623	1532	1555	1494.6	108.578543	7.264722534
Point 5 (𝜇m)	1360	1184	1474	1303	1373	1338.8	106.2341753	7.935029526
Mean (𝜇m)	1403	1378	1540	1546	1576	1484.6 *
SD † (𝜇m)	97.49	201.9	158.9	141.5	136.7		159.6 *
CV ‡ (%)	6.95	14.65	10.32	9.16	8.67			10.8 *

^† Standard deviation;

^‡ Coefficient of variation (SD ÷ Mean × 100);

^*value from overall 25 data

To explain the discrepancy between the ratio of both instruments’ internal diameter (14%) and histological width (22.4%) of both samples, the horizontal shrinkage of both groups was analyzed since it is a known artifact of conventional punch biopsy [6, 17, 18, 19, 20, 21]. The result showed that the microcores underwent significantly less horizontal shrinkage (p < 0.0001) than the punch biopsies did in relation to the internal diameter of the instrument used to obtain the biopsy (19.4% vs. 50.5%, respectively) (Figure 8a,b, Table 3).

FIGURE 8.

Graphical comparison of histological width (a) and horizontal shrinkage of the skin samples (b) between the micro‐coring and punch biopsy techniques. The micro‐coring technique demonstrates significantly less variation in the width of the samples as well as horizontal shrinkage (p < 0.0001).

TABLE 3.

Post‐shrinkage width.

	Microcores					3 mm punch biopsy samples
	1	2	3	4	5	1	2	3	4	5
Point 1 (%)	97.3	76.8	90.3	97.6	96.6	50.3	56.5	58.5	51.5	53.0
Point 2 (%)	78.7	58.1	82.8	76.5	83.3	49.6	47.1	50.4	56.6	58.5
Point 3 (%)	91.3	70.5	78.2	73.4	81.8	42.4	40.8	44.4	51.8	53.5
Point 4 (%)	73.8	72.6	95.6	70.5	83.8	46.2	45.9	54.1	51.1	51.8
Point 5 (%)	72.9	70.2	96.1	73.1	76.8	45.3	39.5	49.1	43.4	45.8
Mean (%)	82.8	69.6	88.6	78.2	84.4	46.8	45.9	51.3	50.9	52.5
SD † (%)	10.9	7.0	7.9	11.0	7.3	3.25	6.73	5.30	4.72	4.56
Mean in Total ‡ (%)	80.7					49.5

^† Standard deviation;

^‡ value from overall 25 data

The measured heights highlighted another discrepancy between the micro‐cores and the 3 mm punch biopsies. Interestingly, the 3 mm punch biopsies were about 2.9 times longer than the micro‐cores despite being taken from the same human abdominal skin sample. This indicates a tissue deformation of the 3 mm punch biopsy rather than overall shrinkage or incomplete sampling of the micro‐cores.

To estimate the pre‐excision thickness of skin under the assumption that tissue deformation occurs without tissue removal or shrinkage, and that both the micro‐core and 3 mm punch biopsy samples yield cylindrical specimens, we can apply the volume formula for a cylinder, $V=\pi {(\frac{d}{2})}^{2}h$. For the micro‐core, with a diameter of 331 µm and a height of 1350 µm, the calculated diameter in the absence of deformation should be 413 µm, leading to an estimated skin thickness of 867 µm. Similarly, for the 3 mm punch biopsy, where the diameter is 1485 µm and the height is 3949 µm, the corresponding diameter without deformation should be 3000 µm, giving an estimated skin thickness of 968 µm.

The estimated skin thickness values are comparable but slightly less than expected for abdominal skin, likely due to tissue shrinkage during histological processing.

3.4. Histological Examination of a Melanocytic Lesion

Our novel method for harvesting and processing microcores was used to obtain samples from a benign melanocytic lesion for histological examination. A dermoscopic examination of this 6 × 4 mm lesion demonstrated the typical features of a benign melanocytic nevus, including a reticular pigment pattern and moderately demarcated border with subtle peripheral fading (Figure 9a–c). One micro‐core was obtained from the lesion's center and another was obtained from the immediate peri‐lesional area. With H&E staining, the microcore of the lesion demonstrates nests of melanocytes within the epidermis arranged along the basal layer in rete pegs beneath the intact stratum corneum without cellular atypia or dermal invasion, and scattered dermal melanosome drops and suspicious melanophages in the superficial dermis (Figure 9d). Immunohistochemical staining highlights one of the rete pegs, demonstrating grouped S‐100‐positive melanocytes, supporting the diagnosis (Figure 9e). The peri‐lesional micro‐core demonstrated no significant histopathological findings (Figure 9f). Overall, the histological examination revealed the typical histological features of a benign junctional nevus.

FIGURE 9.

Dermoscopy image of a benign melanocytic lesion on abdominal skin obtained from abdominoplasty (a). The lesion exhibits an oval shape measuring 6 × 4 mm, characterized by a dark brown coloration indicative of a typical reticular pattern observed in benign melanocytic nevi. The lesion displays a moderately demarcated border with subtle fading. Pigment density appears to be more pronounced at the center, gradually tapering toward the periphery. There is an absence of vascularity within the lesion. A micro‐core biopsy was taken from the lesion's center (white arrow, b) and in the immediately adjacent perilesional area (black arrowhead, c). H&E staining of the sample from the lesion center reveals nests of melanocytes arranged along the basal layer in rete pegs in the epidermis beneath the intact stratum corneum without cytologic atypia or dermal invasion (d). Within the superficial dermis, scattered dermal melanosome drops and suspicious melanophages are noted. Immunohistochemical staining with S‐100 on the same biopsy specimen highlights one of the rete pegs, showing S‐100‐positive melanocytes (e). The perilesional sample showed no noticeable histopathological findings (f). (Scale bar = 2 mm for a–c and 25 𝜇m d–f).

4. Discussion

This study shows that the micro‐coring biopsy technique using hypodermic needle modified to have two prongs at the tip and holes on the side wall can be used to obtain histological specimens. The technique showed a streamlined process yielding stainable columnar skin samples comparable to the conventional punch biopsy technique. Small‐diameter columnar biopsies could be beneficial since the novel apparatus used in our study resulted in a significantly smaller wound compared to punch biopsies and the size 22‐gauge needle used for our experiments has an internal diameter shown to be below the scarring threshold [13, 22].

There are two key structural features of the novel apparatus that minimize physical manipulations of the sample tissue compared to the 3 mm punch: the two pronged tip and side holes. Although punch instruments require a repetitive rotational motion for cutting, the micro‐coring needle can initiate the surface penetration with two points at the tip, minimizing the shear stress of the sample. Punch biopsies also require a pinching manipulation of the tissue with forceps at least once, which increases the probability of deformation and crush artifact once more [23]. Although minimizing forceps manipulation by extracting the punch apparatus from the skin without twisting or tilting may seem like a solution [24], it often leads to uneven tissue thickness due to uneven sheer stress along the depth. The tissue sampled with micro‐coring needle does not meet any pinching instruments until the tissue undergoes the embedding step since the side holes enable solution penetration and sample immersion for tissue fixation and processing inside the apparatus. Thus, the novel apparatus not only results in smaller wounds, but eliminates handling of the tissue in its pre‐fixed and pre‐processed state, when it is the most fragile.

The value and importance of the holes in the side wall of the needle is demonstrated by the resulting samples that underwent the same processing technique with needles without the holes in the side wall as well as samples from the needle which were removed immediately instead of undergoing fixation and processing inside the needle yielded inferior histological results. The samples from the needles with the holes in the side wall note only look better histologically, but were significantly easier to work with, likely due to increased sample stability inside the needle from the friction the holes provide [7]. Removal of the un‐fixed and un‐processed micro‐core from the needle caused it to crush and break. Although there might be solutions to improve handling of the micro‐core outside of the needles, several attempts on our side such as removal via pressured air or flow of liquids for removal remained unsuccessful due to sample size and handling issues. Moreover, having the clinical staff to process the micro‐core outside the needle, rather than leaving it fixed within the needle for a histology technician to handle, would add extra steps for the clinical team, potentially obstructing broader clinical adoption. This highlights the importance of the fixation and processing inside the needle, and the importance of the holes in the needle side wall to complete these steps.

Beyond the benefit of minimal tissue handling, this novel technique can be implemented into any standard tissue processing setting because the reagents and machines are readily available and the typical histology workflow is not compromised. In fact, the efficiency may even be enhanced since smaller tissue samples require less processing time. This technique is also ideal for patients because the micro‐wounds do not require sutures, and heal faster than larger wounds, as there is less area for re‐epithelialization [25], and minimize the risk of scarring. In clinical practice, a technique that does not require sutures or tissue manipulation with instruments to capture and place in a sample jar will improve efficiency of biopsies and reduce the burden on the physician.

The length discrepancy between the micro‐cores and the 3 mm punch biopsies can be explained by the difference in horizontal shrinkage of each type of biopsy. After accounting for skin deformation, the resulting skin thickness measurements were comparable between the two biopsy methods. This finding is significant as it underscores the greater deformation associated with larger biopsies. Despite being substantially shorter than 3 mm punch biopsies, micro‐core biopsies represent full‐thickness samples and more accurately reflect the true skin structure compared to larger biopsies.

It should be noted that a smaller diameter of sampled tissue implies an increased risk of sampling error [23, 26, 27]. This becomes particularly crucial in suspicious pigmented lesions, where complete excisional biopsy is recommended but may not always be feasible [27]. In such cases, using the novel apparatus would result in unavoidable partial biopsy, with the associated risk of sampling error, which can be problematic. However, in certain clinical scenarios, this new biopsy method may be helpful. These scenarios involve relatively homogenous skin lesions, especially in exposed areas of the skin where it is important to minimize scarring. For example, common papular lesions on the nose that look similar to each other including basal cell carcinomas, sebaceous hyperplasia, fibrous papule of the nose, and intradermal nevus. Other examples could be vitiligo versus pityriasis alba, xanthelasma versus syringoma, melasma versus ochronosis, and freckles versus Hori nevus, among many others. Each of these diagnoses vary in terms of treatment and prognosis, so a biopsy‐confirmed diagnosis would be useful in guiding the treatment regimen and patient counseling. Moreover, in many inflammatory skin diseases where the skin is fragile and multiple site biopsies are required, this method could be convenient. For instance, in cases such as bullous diseases where immunofluorescence studies are necessary on the multiple perilesional site, or in inflammatory skin conditions like lupus erythematosus or vasculitis. Due to its relatively low invasiveness, it could offer a convenient option for improved post‐biopsy management and outcomes.

Incorporating dermoscopy into the procedure is important to visually record the lesion itself as well as the precise locations of the sampled sites, as demonstrated in our study, may enable location‐recorded focally diffuse multiple‐site biopsies, which would be challenging to do with conventional punch biopsies.

Certainly, regarding the examples mentioned above, there is still a degree of uncertainty due to the lack of clinical use, as the skin used in our study was ex vivo tissue. The importance of further in vivo testing to confirm he practicality of this technique in a standard clinical setting is crucial, especially for testing its usefulness in analyzing atypical or larger skin lesions. Although this small‐sized biopsy is expected to be advantageous in patient tolerability, recovery, and aesthetic outcome, it has not been clinically confirmed. Therefore, further clinical studies are expected utilizing broader spectrum of micro‐coring needle specifications. It is reasonable to expect that topical anesthesia, at times with supplemental local lidocaine injection depending on patient sensitivity and the target area, would be used for live patients, as is used for the already introduced minimally invasive micro‐coring system for skin rejuvenation [12, 13].

5. Conclusion

Here, we demonstrate a novel technique for a skin micro‐biopsy using a simply modified hypodermic needle that allows fixation and processing of the tissue inside the sampling instrument that yields small, full‐thickness columnar skin histological samples with minimal post‐sampling deformation and risk of scarring. Further clinical studies are expected to confirm the diagnostic ability of this technique for certain dermatological conditions and reduced post‐biopsy issues from the procedure. We hope that this work will furnish clinicians with more advanced diagnostic modalities, fostering progress in the field.

Funding

The authors have nothing to report.

Ethics Statement

This study was conducted in accordance with the ethical standards of the Mass General Brigham Institutional Review Board.

Conflicts of Interest

Elisabeth Roider is a shareholder and founder of Maximon AG and its holding ventures and a founder of EMR BioHealth GmbH. Elisabeth Roider receives funding from the Goldschmidt Jacobson Foundation and the Swiss National Science Foundation and has received consultant and/or speaking fees from Eli Lilly, Galderma and Incyte. Dieter Manstein has contracted research with Shiseido and PIG Photonics. Dieter Manstein's hospital employer, Massachusetts General Hospital, has licensing contracts with various companies including Abbvie (Zeltic), Cytrellis, and Candela. Dieter Manstein receives consulting fees from Avava and R2. Dieter Manstein is the owner/co‐owner of Blossom Innovations and GME German Medical Engineering. William G. Austen receives royalties from Cytrellis, Biosystems, and Durvena. William G. Austen is an advisor for Cytrellis, Biosystems, Durvena, and Sientra. Ga Ram Ahn, Dieter Manstein, and Rebecca Lapides have filed a provisional patent for method and apparatus for microcore tissue sampling and tissue analysis. All other authors declare no competing interests.

Data Availability Statement

The data that support the findings of this study are provided. Additional information is available from the corresponding author upon reasonable request.