WO2025212805A1 - Method and apparatus for microcore tissue sampling and tissue analysis - Google Patents

Abstract

Methods and systems are provided for obtaining microcored tissue samples from a living subject that minimize scarring while providing samples suitable for various tissue analyses. A hollow microcoring needle is provided having a diameter less than about 1 mm, a tip configured to pierce skin or other tissue, and a plurality of holes or openings along the needle walls to facilitate exposure of the tissue within the needle to various substances. A needle containing an extracted tissue sample can be placed in a fixating solution such as a 10% buffered formalin solution, then processed to infiltrate the tissue with paraffin. The fixated tissue is removed from the needle by pushing a wire through the needle core to eject it. The tissue can optionally be treated with various substances while encased within the needle prior to fixation and further processing.

Description

TITLE

METHOD AND APPARATUS FOR MICROCORE TISSUE SAMPLING AND TISSUE ANALYSIS

CROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] The present application relates to and claims priority from U.S. Provisional Patent Application Serial No. 63/573,401 filed April 2, 2024, the disclosure of which is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

[0002] The present disclosure relates to methods and apparatus for obtaining small (“microcore”) tissue samples within a hollow needle, where the needle is smaller than conventional punch biopsy needles, thus reducing scarring and damage to the patient. The sampling needles can be configured with various hole or opening configurations to facilitate fixation of the tissue within the needle and/or access to the tissue by incubator media, analytical instruments, etc.

BACKGROUND

[0003] Punch biopsies are considered a primary technique to obtain skin specimens that are full-thickness and that can be imaged in order to make dermatologic diagnoses. Disposable or sterilizable punch instruments can be obtained of various sizes. Punch biopsies of ~2-4 mm in diameter, often about 3 mm, are the commonly used sizes in order to obtain adequate diagnostic information from the specimen. This size range allows for visualization and accurate diagnosis of a multitude of skin pathologies, while reducing the extent of injury or wounding to the patient or subject. [0004] While punch biopsies have been the standard of care for making accurate dermatologic diagnoses for many decades, they do have certain limitations. Punch biopsies create circular wounds in the skin. To minimize scarring and other complications, careful suturing, appropriate dressing, and close observation is required. However, despite taking extreme caution, complete healing without any noticeable scarring is difficult to achieve. Noticeable scarnng can reduce patient satisfaction, as such scars are generally regarded as unsightly. As a result, in situations with a low risk of significant harm based on a benign list of differential diagnoses, clinicians and patients often hesitate to perform the biopsy, opting for other ways to narrow the differential such as a therapeutic trial, resulting in a more gradual process to establish the diagnosis. Finally, punch biopsies are susceptible to biopsy artifact that occurs once the specimens are removed from the skin as a result of tissue shrinkage.

[0005] For these reasons, there have been several experimental attempts to implement skin micro-biopsy techniques. For example, a lancet-like microbiopsy has been used for safe and successful skin microbiopsy sampling for patients with Epidermolysis Bullosa Simplex. However, these samples were too small to show full-thickness skin. Several other studies have utilized micro-punch biopsies to obtain full-thickness skin samples. However, they have not been w idely used and the feasibility of tissue processing after sample acquisition is not clear and has not been w ell-described.

[0006] A microcoring system has been described that utilizes a hy podermic needle or the like with a two-prong tip created by cutting and shaping the needle on two sides. This system has been used to remove full-thickness skin micro-biopsies without scarring for skin tightening and rejuvenation purposes and is described in, e.g., Pozner JN, Kilmer SL, Geronemus RG, Jack M, Bums JA, Kaminer MS. Cy trellis: A Novel Microcoring Technology⁷ for Scarless Skin Removal: Summary⁷ of Three Prospective Clinical Trials. Plast Reconstr Surg Glob Open. 2021 Oct;9(10):e3905. Although the system is simple enough to automatically harvest full-thickness microcores from skin and is being widely used, systems and methods for histological analysis and other treatments of such microcores that are taken out from the donor site are lacking.

SUMMARY

[0007] Embodiments of the disclosure can provide a method for obtaining a tissue sample for analysis using a hollow microcoring needle that is much smaller than conventional biopsy punches, thereby leading to reduced tissue disruption and scarring. The needle has an outside diameter less than 1 mm and a distal end of the needle can be provided with a tip configured to pierce tissue of a subject, such as the skin. A plurality of holes can be provided in the wall of the needle, where the width of each hole is preferably between 1/3 and 2/3 of the outside diameter of the needle. The method includes advancing the needle into the tissue of the subject at a particular location where the tissue sample is to be removed. The needle can be inserted to a particular depth to trap the tissue sample within the hollow core of the needle. The needle can then be retracted from the tissue to remove the tissue sample from the subject.

[0008] The tissue sample can then be fixated while it remains within the needle. Such fixation can be achieved, e.g., by immersing the needle containing the tissue sample into a fixating solution, such as a 10% solution of buffered formalin. Other fixating solutions may also be used in further embodiments.

[0009] The tissue sample can then be stabilized while inside the needle by infiltrating it with paraffin (or a comparable substance) while it remains within the needle. This stabilization can be achieved, e.g., by placing the needle containing the fixated tissue sample in a cassette, and placing the cassette in a processing arrangement to infiltrate the tissue sample with paraffin. [0010] After stabilization, the tissue sample can be removed from the needle by pushing a wire through the hollow core of the needle to eject the sample. The wire can have a diameter that is approximately the same as the inside diameter of the needle, to effectively remove the stabilized tissue sample while minimizing undesirable deformation or structural modifications to the sample. The ejected tissue sample can then be embedded in a substance, such as paraffin, to facilitate slicing of the embedded sample for visual observation and analysis of the sample. The slicing can be performed using a conventional microtome or the like.

[0011] In further embodiments, the needle containing the tissue sample can be placed in an incubator prior to fixating it, to maintain viability' of the sample for a period of time. The media provided in the incubator can include one or more substances selected to affect the tissue sample prior to fixating and stabilizing the sample for analysis. The substances can include one or more of an androgen, an immunomodulatory drug, an anti-inflammatory drug, an antifungal drug, an antiviral drug, or a bacterium.

[0012] In additional embodiments, a plurality of samples can optionally be obtained through the plurality of holes in the needle containing the tissue sample, thereby facilitating obtaining small tissue samples from a variety of depths within the tissue. In still further embodiments, at least two holes in the needle wall can be diametrically opposed, to facilitate performing an optical analysis of the tissue sample through the diametrically-opposed holes while the tissue sample remains within the needle.

[0013] In other embodiments of the disclosure, a microcoring needle for obtaining a tissue sample for analysis can be provided. The hollow microcoring needle can have an outside diameter less than 1 mm, and a distal end of the needle can be provided with a tip configured to pierce tissue of a subject, such as the skin. A plurality of holes can be provided in the wall of the needle, where the width of each hole is preferably between 1/3 and 2/3 of the outside diameter of the needle. The distance between adjacent holes is preferably greater than the width or diameter of the holes, to support structural integrity of both the needle and the tissue sample within. In still further embodiments, at least two holes of the plurality of holes can be formed diametrically opposed on the wall of the needle. This configuration can facilitate optical analysis of the tissue sample through the opposing holes while the tissue sample remains undisturbed within the hollow needle. Such optical analysis may be performed before or after fixation and/or stabilization of the tissue sample within the needle.

BRIEF DESCRIPTION OF DRAWINGS

[0014] Further objects, features and advantages of the present disclosure will become apparent from the following detailed description taken in conjunction with the accompanying figures showing illustrative embodiments, results and/or features of the exemplary embodiments of the present disclosure, in which:

[0015] FIG. 1 A is an image of an exemplary pronged microneedle having a plurality of holes in the wall according to one embodiment of the disclosure;

[0016] FIG. IB is a different view of the microcoring needle shown in FIG. 1 A;

[0017] FIG. 1C is yet another view of the microcoring needle shown in FIG. 1A;

[0018] FIG. ID shows a conventional punch biopsy instrument at the same magnification of the microneedle show n in FIGS. 1A-1C;

[0019] FIG. 2 illustrates an exemplary procedure for harvesting and treating tissue samples in accordance with certain embodiments of the disclosure;

[0020] FIG. 3A shows a tissue processing cassette with microcoring needles containing tissue samples being placed therein for further processing; [0021] FIG. 3B shows the tissue processing cassette of FIG. 3 A that is loaded and ready for processing of the tissue samples contained therein;

[0022] FIG. 3C shows a microcore tissue sample being removed from the needle using a guide wire after the tissue sample has been processed and fixated using the cassette shown in FIGS. 3A-3B;

[0023] FIG. 3D is an image of three microcore tissue samples after they have been fixated and removed from microcoring needles as shown in FIG. 3C;

[0024] FIG. 4A is an image of a microcore sample after it has been fixated, removed from the microcoring needle, sectioned, and stained with H&E (hematoxylin and eosin) dyes, showing the full-thickness nature of the microcore samples, with a scale bar that is 250 /rm wide;

[0025] FIG. 4B is an image of another microcore sample similar to the one shown in FIG. 4A, with a scale bar that is 250 /rm wide;

[0026] FIG. 4C is an image of still another microcore sample similar to the one shown in FIG. 4A, with a scale bar that is 250 /zm wide;

[0027] FIG. 4D is an image of yet another microcore sample similar to the one shown in FIG. 4 A, with a scale bar that is 250 rm wide;

[0028] FIG. 4E is a magnified image of the upper part of a microcore sample similar to those shown in FIGS. 4A-4D, containing epidermal tissue, with a scale bar that is 50 /zm wide;

[0029] FIG. 4F is a magnified image of a central portion of a microcore sample similar to those shown in FIGS. 4A-4D, showing horizontally oriented collagen bundles with several fibroblasts and a segment of the eccrine duct, with a scale bar that is 50 /rm wide; [0030] FIG. 4G is a magnified image of a lower portion of a microcore sample similar to those shown in FIGS. 4A-4D, showing a part of the subcutaneous fat layer at the lower border of the reticular dermis, with a scale bar that is 50 /zm wide;

[0031] FIG. 5A is an image of an upper portion of a microcore sample after it has been fixated, removed from the microcoring needle, sectioned, and stained with periodic acid-Schiff (PAS) stain, with a scale bar that is 50 /rm wide;

[0032] FIG. 5B is an image of a microcore sample after it has been fixated, removed from the microcoring needle, sectioned, and stained with Masson’s trichrome stain, with a scale bar that is 50 zm wide;

[0033] FIG. 5C is an image of a microcore sample after it has been fixated, removed from the microcoring needle, sectioned, and stained with Verhoeff van Gieson elastin stain, with a scale bar that is 50 /zm wide;

[0034] FIG. 6A shows a size comparison of wounds formed using a microcoring needle as described herein (left) and a conventional punch biopsy (right), with a 1 mm scale bar;

[0035] FIG. 6B show s a size comparison of a microcoring needle as described herein (left) and a conventional punch biopsy (right), with a 1 mm scale bar;

[0036] FIG. 6C shows horizontal measurements made on a section of a microcore sample to determine lateral shrinkage, with a scale bar of 500 /zm;

[0037] FIG. 6D shows horizontal measurements made on a section of a punch biopsy sample to determine lateral shrinkage, with a scale bar of 500 /zm;

[0038] FIG. 7A is a plot showing width measurements made on 5 fixated samples of both microcore needle samples and punch biopsy samples, compared to the original sample widths (internal diameter of the needle/punch); [0039] FIG. 7B is a tissue sample shrinkage plot showing fixated sample widths as a percentage of the original (needle/punch) width for the width measurements shown in the plot of FIG. 7 A;

[0040] FIG. 8A is a dermoscopic image of a 6 x 4 mm benign melanocytic lesion obtained from abdominoplasty, with a 2 mm scale bar;

[0041] FIG. 8B is a dermoscopic image of the lesion shown in FIG. 8A, with a white arrow- indicating the location where microcoring biopsy samples were obtained from the center of the lesion, with a 2 mm scale bar;

[0042] FIG. 8C is a dermoscopic image of the lesion shown in FIG. 8A, with a black arrow indicating the location where microcoring biopsy samples were obtained from the adjacent perilesional area of the lesion, with a 2 mm scale bar;

[0043] FIG. 8D is an image of a microcore sample obtained from the center of the lesion (white arrow) shown in FIG. 8B, stained with an Immunohistochemical stain, with a 25 /rm scale bar;

[0044] FIG. 8E is an image of an H&E-stained microcore sample obtained from the center of the lesion (white arrow) shown in FIG. 8B, with a 25 m scale bar;

[0045] FIG. 8F is an image of a microcore sample obtained from the periphery of the lesion (black arrow) shown in FIG. 8C, stained with an Immunohistochemical stain, with a 25 ftm scale bar; and

[0046] FIG. 9 illustrates the 2-stage snare grasper device shown in FIG. ID with the snare wire retracted, prior to retraction of the grasper end.

[0047] While the present disclosure will now be described in detail with reference to the figures, it is done so in connection with the illustrative embodiments and is not limited by the particular embodiments illustrated in the figures. It is intended that changes and modifications can be made to the described embodiments without departing from the true scope and spirit of the present disclosure as defined by the appended claims. It is also noted that any trademarks, trade names, or other proprietary identifiers shown in the figures are for illustrative purposes only, and do not imply any affiliation or agreement between the owners of such identifiers and the Applicant submitting the present disclosure.

DETAILED DESCRIPTION

[0048] Embodiments of the present disclosure provide novel systems and techniques for utilizing a modification of a two-prong hypodermic needle that enables an easy extraction of full-thickness skin microcore samples from whole tissue, and further facilitates histological analysis and a streamlined post-sampling process of such microcore tissue samples, including improved ability to maintain viable tissue samples for a longer time after their removal from a subject.

[0049] Embodiments of the disclosure employ modified microcoring needles to facilitate histological analysis and other processing procedures on the tissue samples obtained. In one exemplary embodiment, a 20-gauge hypodermic needle approximately 12 mm in length was positioned inside a rotary' tool and a translation stage was used to advance the needle towards a diamond cutting disc until the cutting disc reached the midline of the needle. A 20- gauge needle has an outside diameter of about 0.9 mm, and an inside diameter of about 0.6 mm. 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 4 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 easily through the skin vertically to obtain microcore tissue samples.

[0050] In one embodiment, a microcoring needle capable of allowing thorough fixation of tissue while inside the needle was formed as above that further includes 4 parallel, staggered rows of 4 holes into the needle, with the first hole aligned to one of the pointed tips. Each hole was 0.4 mm in diameter with a pitch of 0.5 mm and a nominal angle for the tip of 12°. This exemplary coring needle configuration is shown in FIGS. 1A-1C. The presence of these holes enables, inter alia, fixative solution to penetrate the needle and immerse the skin specimen inside the needle to allow for efficient fixation of the tissue. The number, size, and spacing of such holes can be varied in further embodiments as described below. In general, it is preferable that a distance between adjacent holes is greater than the diameter or width of the holes, to maintain structural integrity of the needle and maintain integrity of the tissue sample within the needle.

[0051] In an exemplar}' procedure, human skin specimens were obtained from the excess tissues removed during elective abdominoplasties. The human tissue samples would otherwise have been discarded and were obtained without any identifying information besides age and sex of the patient. The procedure described below provides a comparison betw een a conventional punch biopsy needle and a microcoring needle according to exemplary embodiments of the present disclosure.

[0052] The skin specimens were prepared by removing as much excess subcutaneous fat tissue as possible, leaving only a thin layer behind for retrieval within the biopsy specimens. The surface of the skin was gently cleaned with an alcohol wipe to remove excess blood and debris on the surface. The skin was pulled taught to remove wrinkles and the exemplary' 20 gauge microcore needle described above was positioned perpendicularly on the surface of the skin and driven vertically through the skin through the epidermis, dermis, and subcutaneous tissue as quickly as possible to effect penetration of the skin tissue. The needle was then very slowly removed from the skin to avoid disrupting the sample inside. Microcoring needles with and without holes were used to harvest samples so that the samples could be compared between the two types of needles.

[0053] With the microcore skin samples still inside both types of the microcoring needles (with and without side holes), the needles were placed into 10% buffered formalin and fixated in this solution overnight at room temperature. Then, each needle with the sample still inside was placed inside a cassette and processed in a tissue processing machine such that it was completely infiltrated with suitable paraffin wax. Next, a guide wire slightly narrower than the internal diameter of the needle (603 pm) was used to remove the fixated and processed microcore from each of the needles. The microcores were then embedded in paraffin wax and a microtome was used to cut 5pm thick sections. The sections were mounted onto Poly-L-Lysine glass slides. The detailed scheme depicting the methodology used to harvest, process in paraffin, and section the microcore samples is illustrated in FIG. 2.

[0054] Conventional 3 mm punch biopsies were also taken from the skin using standard methodology for taking punch biopsies. Briefly, the punch biopsy instrument (shown in FIG. ID) was positioned vertically on the skin and rotated both clockwise and counterclockwise using slight downward pressure and a twirling motion until the punch biopsy instrument penetrated the skin layers.

[0055] The 3 mm punch biopsies were removed from the punch biopsy instrument and placed into a tissue processing cassette, which was submerged in 10% buffered formalin and fixated in this solution overnight. Next, the punch biopsy samples were processed, embedded in paraffin wax, and sectioned at 5pm thick, similar to the microcore samples. The sections were mounted onto Poly-L-Lysine glass slides. [0056] Both the microcores and the 3 mm punch biopsy specimens were stained with hematoxylin and eosin (H&E) dyes as well as several representative special stains commonly used in dermatology. These stains included the Verhoeff Van Gieson elastin stain, periodic acid-Schiff stain (PAS), Masson Tri chrome stain, and the SI 00 protein stain. Standard protocols described in the data sheets accompanying the kits used for each of the special stains were used.

[0057] The design of the microcoring needle having a 2-prong tip and holes in the sidewall, described herein and shown in FIGS. 1A-1C, enabled fixation and processing of skin samples inside the needle by placing this entire apparatus into the tissue processing cassette, which is shown in FIGS. 3A and 3B. Using this microcoring needle, human adult abdominal skin samples were reliably collected, fixated, processed, and removed from the needle with a guide wire (shown in FIG. 3C), yielding rectangular columns of skin (shown in FIG. 3D). The skin samples readily underwent sectioning and staining with H&E dyes, demonstrating the full-thickness nature of the skin microcores (FIGS. 4A-4D).

[0058] Sections from the microcoring samples that underwent hematoxylin and eosin (H&E) staining demonstrate a consistent rectangular or cylindrical shape, reflecting the appearance of the processed samples observed during the embedding process (see FIGS. 4A- 4D). In the upper part of the samples (as shown, e.g., in FIG. 4E). a typical basket-weave pattern of the stratum comeum and stratified epidermal layer including the stratum granulosum are observed. Sparse immune cells and superficial vessels are also present in the upper dermis. In the mid-part of the samples (shown, e.g., in FIG. 4F). compact horizontally oriented collagen bundles with several fibroblasts and a segment of the eccrine duct were observed. At the lower part of the samples (shown, e.g., in FIG. 4G), a part of subcutaneous fat layer is shown at the lower border of the reticular dermis. In these figures, the scale bars in FIGS. 4A- 4D are 250 qm in width, and are 50 m in width in FIGS. 4E-4G.

[0059] The microcores demonstrate a consistent rectangular or cylindrical shape (FIGS. 4A-4D), reflecting the appearance of the processed samples when they removed from the microcoring needle, just prior to embedding (shown in FIGS. 3C-3D). Histologically, the microcores demonstrate the typical features of the epidermis, including a typical basket-weave pattern to the stratum comeum and a visible stratum granulosum layer (FIG. 4E). In the upper dermis, sparse immune cells and superficial vessels can be visualized (FIG. 4E). In the middermis, compact, horizontally oriented collaged bundles with several fibroblasts and a segment of the eccrine sweat duct can be observed (FIG. 4F). In the most inferior aspect of the microcore, part of the subcutaneous fat layer can be seen adjacent to the reticular dermis (FIG. 4G).

[0060] To demonstrate the versatility of staining that is possible using microcores obtained from the presently disclosed harvesting and processing techniques, three special stains were performed. The periodic acid-Schiff (PAS) stain, shown in FIG. 5A, highlights the basement membranes of the epidermis and superficial vessels. The Masson’s tri chrome stain, shown in FIG. 5B, reveals dermal collagen fibers in a green hue, differentiating them from other components stained in red. The Verhoeff van Gieson elastin stain, shown in FIG. 5C, demonstrates elastin fibers in black with collagen stained in a magenta color. Each of these special stains applied to the microcore samples exhibits distinct and distinguishable results, which can be used for improved assessment of the tissue architecture for diagnostic purposes.

[0061] Microcoring biopsy sections were stained with periodic acid-Schiff (PAS), Masson's trichrome, and Verhoeff van Gieson staining (shown in FIGS. 5A, 5B, and 5C, respectively). PAS staining (FIG. 5A) highlighted the basement membranes of the epidermis and superficial vessels. Masson's trichrome staining (FIG. 5B) revealed dermal collagen fibers in a green hue, distinctly differentiating them from other components stained in red. In

Verhoeff van Gieson staining (FIG. 5C), elastin fibers appeared black, while collagen was stained in magenta color, demonstrating their distinct staining properties and composition within the reticular dermis. The length of the scale bar in each of FIGS. 5 A-5C is 50 /im)

[0062] The exemplary microcoring needle used was a standard 20 gauge needle with an internal diameter of 603 pm, approximately 80% smaller in width/diameter than that of the 3 mm punch biopsy instrument it was compared with. Consequently, the wound size created by the microcoring needle is approximately 80% smaller compared to that of the 3 mm punch biopsy, as shown in FIG. 6A. The visual difference in the internal diameters of these two instruments, shown in FIG. 6B, can be easily appreciated. The length of the scale bars in FIGS. 6 A and 6B are 1 mm. The reduced size of the wound formed by the microcore needle as compared to that formed by the conventional biopsy needle (FIG. 6A) indicates the desirability of using microcoring needles to obtain tissue samples, if such smaller samples are useful and sufficient for analysis.

[0063] As horizontal shrinkage is a known artifact of punch biopsies, a comparison of horizontal shrinkage between the microcore samples and punch biopsy samples was performed. Horizontal or lateral measurements were made on sections of 5 microcore samples (one such sample is shown, e.g., in FIG. 6C) and five 3 mm punch biopsies (one such sample is shown, e.g., in FIG. 6D). The lengths of the scale bars in FIGS. 6C and 6D are 500 /im. These horizontal sample measurements were plotted on a graph, shown in FIG. 7A, and compared to determine the histological width of the different samples and to estimate the horizontal shrinkage that samples undergo after removal from the skin. The microcore samples demonstrated significantly less variation in histological width compared to the punch biopsies (as shown in FIG. 7A). Further, the microcore samples underwent significantly less horizontal shrinkage, expressed as a percentage of the original width (e.g., internal diameter of the sampling instrument) than the punch biopsy samples did (as shown in FIG. 7A).

[0064] In addition to microcoring needles yielding skin biopsy samples that are reliably imageable for diagnostic use, these samples also undergo far less horizontal shrinkage compared to punch biopsies. Horizontal shrinkage is a well-known artifact of punch biopsies. This phenomenon has important implications, including discrepancies between the surgical and pathological reported specimen dimensions, which affects the accuracy of surgical margins reported after excision of a malignant lesion. The examples described herein demonstrate that microcore samples shrink significantly less than 3 mm punch biopsy lesions (see, e g., FIGS. 7A-7B) and consistently demonstrate a rectangular column of skin tissue (see, e.g., FIGS. 4A- 4D). These observations suggest that obtaining and processing microcore samples using the presently disclosed apparatus and technique results in better maintenance of shape and structural integrity of the resulting skin sample compared to the 3 mm punch biopsy. Further, only one punch biopsy is ty pically taken of a skin lesion, which then undergoes shrinkage and manipulation during the imaging process, resulting in a high probability' that the initial orientation in the skin of the specimen removed by the punch will be lost. In contrast, embodiments of the present disclosure facilitate obtaining multiple lesional and perilesional microcore samples from the same lesion, with reduced damage and trauma to the patient as w ell as reduced scarring. Depending on their size, microcore samples can be used to provide specific information about a particular area or margin pertaining to a skin lesion, which is targeted to the precise area that the microcore samples were removed from. With preservation of tissue integrity, reduced shrinkage, and targeted information to specific areas of skin lesions, embodiments of the present disclosure that allow for obtaining microcore samples certainly provide samples with high diagnostic value. [0065] In another demonstrative example based on embodiments of the present disclosure, microcore samples were collected from a benign melanocytic lesion (obtained from abdominoplasty) for definitive diagnosis. Dermoscopy of this 6 x 4 mm lesion, shown in FIGS. 8A-8C, exhibited typical features of benign melanocytic nevi, including a reticular pigment pattern, a moderately demarcated border, and subtle fading, with pigment density appearing more pronounced at the center and gradually tapering towards the periphery. There is an absence of vascularity within the lesion.

[0066] Microcoring biopsy samples were obtained from the lesion's center (white arrow, FIG. 8B) and in the immediately adjacent perilesional area (black arrow, FIG. 8C). The length of the scale bars in FIGS. 8A-8C is 2 mm. With appropriate labeling, it is simple to identify where each microcore sample was taken from and glean specific histological information about that particular area.

[0067] With H&E staining, the microcore sample of the lesion exhibited classic features consistent with a junctional nevus, such as nests of melanocytes within the epidermis, arranged along the basal layer in rete pegs beneath the intact stratum comeum without cellular atypia or dermal invasion, and scattered dermal melanosome drops and suspicious melanophages in the superficial dermis. These observed features are shown in FIG. 8D. Immunohistochemical staining highlights one of the rete pegs, shown in FIG. 8E. demonstrating S- 100-positive melanocytes and supporting the diagnosis. In contrast, the perilesional microcore sample, shown in FIG. 8F, demonstrated no significant histopathological findings. The length of the scale bars in FIGS. 8D-8F is 25 /rm. Overall, the histological results obtained using the microcoring methods and apparatus are consistent with a diagnosis of a benign junctional nevus, and could be used for such diagnosis.

[0068] Staining w ith H&E is the most commonly used technique to visualize basic tissue architecture in skin samples. Tn order for a sample to be effectively stained and used for diagnosis, there should be adequate staining intensity, sufficient contrast between different tissue components, uniformity across the entire tissue section, preservation of tissue morphology, and absence of artifacts. With these criteria met, there is a large spectrum of colors and patterns that can be observed and used for diagnosis of many conditions. Punch biopsies reliably yield samples that meet these criteria. However, with the microcoring apparatus and methods described herein, the microcore samples also meet these criteria for effective staining (see, e.g., FIGS. 4A-4D), demonstrating that they adequately show the important structures that must be visualized for diagnosis of skin conditions while mitigating the invasiveness and risk of scarring associated with punch biopsies, as discussed above.

[0069] For example, in the exemplar}⁷ procedures described herein, the important histological features may be visualized from microcore samples, which include a stratified epidermis and rete ridges along the dermal-epidermal junction (FIG. 4E), human dermal skin elements including fibroblasts, collagen bundles, and adnexal structures (FIG. 4F), and subcutaneous fat at the border of the reticular dermis (FIG. 4G). In contrast, prior uses of microneedle biopsy devices and procedures for harvesting microcore samples involve removing the specimen from the needle prior to fixation and processing, which risks losing and/or damaging the specimen. Embodiments of the present disclosure, which can be used for fixating and processing microcore specimens inside the needle, provide a simple and effective method to process and image microcore samples with 100% reliability. That is, every sample harvested using the disclosed method and apparatus was effectively processed and useable for high-quality staining and imaging. This simple and standardized protocol can easily be performed successfully in any laboratory setting.

[0070] In further embodiments, the needle gauge, hole sizes, number of holes, hole pitch, and configuration of the rows of holes may be varied. For example, hollow needles having different diameters or gauges can be used in embodiments of the disclosure. The needle sizes should be selected such that the inner diameter is large enough to obtain useful samples of tissue, while being smaller than conventional punch biopsy devices (e.g., less than about 1 mm) to minimize or eliminate scarring and local damage.

[0071] Using hypodermic microcoring needles with a modified 2-prong tip that are perforated with holes (see FIGS. 1 A-1C) enables fixation and processing of tissue inside the needle (see. e.g., FIGS. 3A and 3B) because the solutions are able to penetrate the wall of the needle through the holes and immerse the tissue sample therein. The configuration of the exemplary microcoring needles thus minimizes tissue handling and disruption after the samples are removed from the skin. The holes in the side wall of the microcoring needle are an important feature to achieve these advantages, as similar coring techniques using 2-prong microcoring needles without holes resulted in samples that were of too poor quality to meaningfully image.

[0072] In further embodiments, the sizes, shapes, spacings, and/or patterns of holes provided in the microcoring needle can be varied to achieve additional benefits. For example, the hole diameters shown in FIGS. 1A-1C are about half the outside diameter of the needle. Slightly smaller or larger holes can be used, e.g., holes having diameters between about 1/3 and 2/3 the diameter of the needle. Smaller holes can better protect the microcore samples within the needles while still allowing exposure of the sample tissue to various solutions, facilitating the use of various diagnostic tools that access the tissue samples (e.g.. optical analytical tools, described herein below), etc. Larger holes and/or closer hole spacings can be used to increase the exposure of the tissue within the needle to surrounding fluids, provide access to a larger portion of the tissue sample by diagnostic tools, etc. The hole sizes and spacings are generally limited by the need to maintain structural integrity of the needle while extracting microcore samples from bulk tissue.

[0073] In some embodiments, pairs of holes can be provided in the needle that are diametrically opposite each other. This configuration can facilitate the use of optical instruments that can analyze the tissue by passing optical wavelengths through the tissue samples (e.g.. through opposing pairs of holes in the needle). Such optical instruments could entail Spectrophotometry, optical coherence tomography, multiphoton imaging, confocal microscopy, polarized microscopy and surface imaging. These diametrically opposed holes can also be used to extract even smaller sub-samples of tissue (from various tissue depths, e.g., distances along the needle axis) by pushing them out of these opposite holes using a wire or similar tool.

[0074] In further embodiments, elongated openings can be provided in the needle walls to further facilitate access to the microcore samples extracted within the needle. Such openings can have a length along the axis of the needle that is between about 2 and 10 times the hole diameter. For example, an opening can be provided that extends between the two leftmost holes in FIG. 1C, having a width that is substantially the same as the hole diameters. Besides providing increased exposure of the microcore sample in the needle to surrounding solutions and the like, such elongated openings can facilitate analysis of the tissue at different depths (e.g., different distances along the axis of the needle) while the microcore sample remains inside the needle. However, such larger openings may compromise the structural integrity and/or distort the original geometry of the tissue sample when it is fixated within the microcoring needle and subsequently removed as described herein.

[0075] In yet another embodiment, rows of such elongated openings can be provided at different radial locations around the circumference of the needle, where the ends of these openings overlap with respect to distance from the needle tip. This configuration can allow all depths in the microcore sample to be accessed while the sample is contained in the needle to maintain tissue integrity. These ‘'overlapping’’ elongated openings can be provided without corresponding diametrically opposite openings in some embodiments. In further embodiments, they can be provided with corresponding diametrically opposite openings. For example, two sets of diametrically opposed elongated openings, optionally with their ends overlapping with respect to distance from the needle tip. can be provided in a needle, which are located at 90 degrees (or another angle) from each other around the circumference of the needle. This configuration can allow optical instruments to be directed through the sample unimpeded by the needle material, allowing such access along the entire length of the tissue sample within the needle while maintaining structural integrity of the needle.

[0076] In additional embodiments of the disclosure, the tip of the microcoring needle can be shaped to have any one of several other tip configurations configured to pierce skin tissue. For example, five such piercing tip configurations are show n in FIG. 9. These tip shapes are used e.g., in various tissue sampling and aspiration needles. The tip shapes shown in FIG. 9 are commonly referred to as Chiba, Franseen, Westcott, Spinal, and Green tips.

[0077] In additional embodiments of the disclosure, the disclosed needles can be used to obtain microcore biopsy samples from live human skin, and then held in a holder and submerged in media to keep the skin tissue alive inside a conventional incubator environment to undergo additional testing. For example, additional compounds can be added to the media of the incubator, such as certain drugs or compounds, and then after a desired time interval, comparative analyses can be conducted between the microcore samples to determine certain property variations resulting from the different exposures, such as DNA damage. As one example, microcore samples can be obtained from live human skin tissue that was exposed to UV irradiation, and the needles with the microcore samples inside can be immersed inside incubator media. One such needle/sample could be exposed to media with androgens, and another could be exposed to regular media without androgens. After a certain time period, testing of the irradiated tissues exposed to different media can reveal the comparative extent of DNA repair and residual damage in the tissue specimens. The present disclosure provides methods and apparatus for conducting such studies while inducing minimal damage to the patient due to the small size of the microcore needles (e.g., as compared to the size of conventional biopsy punches).

[0078] Various other tissue studies besides assessing the effects of androgens on irradiated tissue can benefit from embodiments of the disclosure. As one example, one tissue specimen can be exposed to a media containing anti-inflammatory drugs, while a corresponding specimen is exposed to regular media without the drugs. Inflammation levels and/or inflammatory markers can be compared between these microcore samples after a desired time has elapsed.

[0079] Similarly, microcore samples of live human skin tissue can be exposed to bacteria or pathogens, and one such tissue specimen can be exposed to antibiotics in the media while leaving another sample untreated with regular media. A comparison can then be made of the relative bacterial loads or presence of pathogens in the treated and untreated specimens to assess the efficacy of the antibiotics.

[0080] In another embodiment, microcore samples of live human skin tissue infected with fungi or viruses can be obtained, and one such tissue specimen can be exposed to antifungal drugs or antiviral drugs in the media, respectively. Such treated microcore samples can be compared to other untreated samples to evaluate the efficacy of the treatments.

[0081] In still another embodiment, microcore samples of live human skin tissue exposed to immune stimuli or triggers can be obtained, with one such sample treated with immunomodulatory drugs in the incubator media while leaving another tissue sample untreated in regular media. Immune response markers and/or cytokine levels can be evaluated in the treated and untreated samples to assess the immunomodulatory effects of the drugs

[0082] These embodiments represent only some examples of how such experiments using needles containing microcore samples placed in incubator environments can provide valuable insights into the efficacy and mechanisms of action of various drugs in treating skin conditions or underlying systemic diseases affecting the skin.

[0083] In yet additional embodiments, microcore samples obtained in accordance with the described methods and apparatus can be used for spatial transcriptomics studies, as taking multiple small microcore samples facilitates the use of multiple samples per analysis, especially when dealing with sensitive areas like the face, without scarring. Samples can be collected from a broad spectrum of patient demographics and conditions to analyze the variability in gene expression. Further, the small size of the microcore samples is ideal for the 11 x 11 mm capture area of the Visium platform, such that analyzing multiple samples at a time can conserve reagents and reduce the cost of such studies.

[0084] Although the invention has been described in terms of particular embodiments and applications, one of ordinary skill in the art, in light of this teaching, can generate additional embodiments and modifications without departing from the spirit of or exceeding the scope of the claimed invention. Accordingly , it is to be understood that the drawings and descriptions herein are proffered by way of example to facilitate comprehension of the invention and should not be construed to limit the scope thereof. It will thus be appreciated that those skilled in the art will be able to devise numerous systems, arrangements and methods which, although not explicitly show n or described herein, embody the principles of the invention and are thus within the spirit and scope of the present invention.

Claims

WHAT IS CLAIMED IS:

1. A method for obtaining a tissue sample for analysis, comprising: providing a hollow microcoring needle, wherein the needle has an outside diameter less than 1 mm and comprises: a tip configured to pierce tissue, and a plurality of holes provided in a wall of the needle, wherein a width of each hole is between 1/3 and 2/3 of the outside diameter; advancing the needle into a tissue of a subject to a particular depth to trap the tissue sample within a hollow core of the needle; retracting the needle from the tissue to remove the tissue sample from the subject; fixating the tissue sample while it remains within the needle; infiltrating the tissue sample with paraffin while it remains within the needle; and removing the tissue sample from the needle by pushing a wire through the hollow core of the needle to eject it.

2. The method of claim 1, further comprising embedding the ejected tissue sample in a substance.

3. The method of claim 1, wherein fixating the tissue sample comprises immersing the needle containing the needle in a 10% solution of buffered formalin.

4. The method of claim 1, wherein infiltrating the tissue sample with paraffin comprises placing the needle containing the fixated tissue sample in a cassette, and placing the cassette in a processing arrangement to infiltrate the tissue sample with paraffin.

5. The method of claim 1, further comprising placing the needle containing the tissue sample in an incubator prior to fixating it.

6. The method of claim 5, wherein a media provided in the incubator comprises a substance selected to affect the tissue sample.

7. The method of claim 6, wherein the substance comprises at least one of an androgen and an immunomodulatory drug.

8. The method of claim 6, wherein the substance comprises at least one of an antiinflammatory drug, an antifungal drug, and an antiviral drug.

9. The method of claim 6, wherein the substance comprises a bacterium.

10. The method of claim 1, further comprising obtaining a plurality of samples through the plurality of holes in the needle containing the tissue sample.

11. The method of claim 1, wherein at least two holes of the plurality of holes are diametrically opposed, and the method further comprises performing an optical analysis of the tissue sample through the diametrically-opposed holes.

12. A microcoring needle for obtaining a tissue sample for analysis, comprising: a hollow microcoring needle having an outside diameter less than 1 mm; a tip of the needle configured to pierce tissue; and a plurality of holes provided in a wall of the needle, wherein a width of each hole is between 1/3 and 2/3 of the outside diameter of the needle.

13. The microcoring needle of claim 12, wherein a distance between adjacent holes is greater than a diameter of the holes.

14. The microcoring needle of claim 12. wherein at least two holes of the plurality of holes are diametrically opposed on the wall of the needle to facilitate optical analysis of the tissue sample.