Methods, tools, and apparatus for cutting and transferring biological materials.

Abstract

In one aspect, the present invention relates to an automated method for transferring biological material (117) from a sample disposed on a planar substrate (115) into a collection tube using a cutting tool (120) having an internal passageway (121) extending between a first and second end of the tool and a breathable filter element (125) spanning the internal passageway, the first end of the tool being provided with an orifice (123) and a scraping blade (124). The method comprises the steps of: a) physically separating biological material from a flat substrate (115) using a scraping blade (124) of a cutting tool; b) during step a), creating a negative pressure at a second end of the tool by connecting the internal passage (121) to a vacuum generator (150) to create an upward air current at the tool orifice (123), which draws the separated material into the internal passage (121), where it is retained under a filter element (125); c) airtightly attaching a collection tube to the exterior of the cutting tool; and d) creating an overpressure at the second end of the tool by connecting the internal passage to a pressure reservoir (160), thereby creating a pressure pulse that expels the biological material retained under the filter element and transports the material (117) into the collection tube.

Description

Technical Field

[0001] The present invention relates to an automated method for cutting a biological material disposed on a flat substrate such as a slide glass and transferring the cut material to a receptacle for further analysis. The present invention further relates to a related apparatus and a disposable tool for use in the apparatus.

Background Art

[0002] For performing molecular analysis of tumors for the purpose of tumor diagnosis, a certain amount and concentration of tumor cells must be present in the sample to be analyzed. Tumor tissue is heterogeneous and contains other tissues and cell types. Thus, the region of interest (ROI) is typically defined as a sample cut from a thin section of tissue placed on a microscope slide. Manual cutting methods are most common, where a laboratory assistant, for example, scrapes material from the ROI using the blade of a surgical scalpel and transfers the scraped material to a collection tube. This requires a high level of skill and there is no guarantee that only the material from the ROI will enter the collection tube, even when performed by a highly skilled laboratory assistant.

[0003] An improved manual method of tissue removal and collection is disclosed in U.S. Patent Application Publication No. 2020 / 038001. This method uses suction to remove manually scraped tissue from a slide. The cutting tool used has three main elements, namely a shaft portion whose one end can be coupled to a suction source, a cap incorporating a blade at one end, and a filter column releasably fixed to the shaft portion via the cap. The filter column comprises a filter member for capturing the cut material sucked into the tool through the cap. After cutting, the filter column is removed from the tool and placed, for example, in a microcentrifuge tube, where the filter member is submerged in a solution for lysing the cut tissue.

[0004] While this method has the advantage of reducing the risk of contamination of tissue samples during the transfer step, the need for a tool that can be disassembled makes the disclosed method and tool unsuitable for use in automated apparatus.

[0005] An example of an automated apparatus for extracting material from biological samples via milling is disclosed in U.S. Patent No. 1,0876,933. The apparatus comprises a head assembly and a stage, the stage for receiving and collecting samples from multiple tissue slides and enabling automated loading and uploading of multiple milling tips into multiple corresponding sample collection vials. During cutting, the head assembly rotates the milling tip while pulling out a plunger, which simultaneously discharges buffer into the blade portion of the milling tool, cuts the tissue, and aspirates the buffer and cut tissue into a designated collection vial by pushing in the plunger. The used milling tip can then be reloaded into a holder to avoid secondary contamination or discarded.

[0006] Laser capture microdissection (LCM) is also known to be used to isolate concentrated populations of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. A method and apparatus for transferring microscopic, isolated specimens, particularly membrane-supported micro-dissectioned specimens, from an object table to an analytical array is disclosed in U.S. Patent No. 8,573,073. The apparatus is equipped with a nano-suction means and comprises a suction tube with a terminal membrane and a vacuum / overpressure unit coupled to the suction tube for aspirating or blowing the specimen into or from the terminal membrane. The apparatus and method of U.S. Patent No. 8,573,073 has the disadvantage of requiring two different tools for dissection and transfer. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] U.S. Patent Application Publication No. 2020 / 038001 [Patent Document 2] U.S. Patent No. 10876933 [Patent Document 3] U.S. Patent No. 8573073 [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] There is still room for improvement in defining simpler methods, tools, and apparatus for cutting and transferring biological materials with minimal contamination, enabling the collection and transfer of materials for automated analysis using the same tools. [Means for solving the problem]

[0009] In a first embodiment, the present invention relates to an automated method for cutting and transferring biological material from a sample placed on a flat substrate, such as a glass slide, into a collection tube, using a cutting tool having an internal passage extending between first and second ends of the tool and a permeable filter element extending across the internal passage. The first end of the tool is provided with an orifice and a scraping blade positioned in the orifice. The method is as follows: a) A step of physically separating the biological material from a flat substrate using a scraping blade of a cutting tool, b) step a) in which a negative pressure is generated at the second end of the cutting tool to generate an upward airflow in the tool orifice, the upward airflow drawing the separated material into the internal passage, in which the material is held below the filter element, c) The step of positioning the collection tube around the orifice so as to make sealing contact with the connection interface in the cutting tool, d) The step of generating an overpressure at the second end of the tool to create an airflow and pressure pulse that discharges the biological material held on the lower side of the filter element and transfers the material into the collection tube, It is equipped with.

[0010] The present invention enables a "dry" method for transferring biological material directly from the slide glass to the collection tube, thereby minimizing the risk of contamination. Furthermore, a single tool can be used to cut, collect, and transfer the biological material, which not only simplifies the configuration of the equipment required to carry out the method in an automated manner but also further reduces the risk of contamination. The method does not utilize any liquids that dilute the biological material or affect the method and quality of molecular analysis of the collected biological material.

[0011] The separation step comprises bringing a scraping blade into contact with a flat substrate and moving a cutting tool toward it. For simplicity, the flat substrate will be referred to below as a glass slide, but as can be understood, any suitable type of substrate on which a biological sample is placed may be used in the method of the present invention.

[0012] Advantageously, the method comprises controlling the relative position of the tool and the slide glass so that the scraping blade engages with the biological material only in the region of interest. This optimizes the quantity and purity of the material collected for analysis.

[0013] The step of appropriately generating negative pressure comprises connecting the internal passage of the cutting tool to a vacuum generator at the second end of the tool, and the step of generating overpressure comprises connecting the internal passage to a pressure reservoir.

[0014] Preferably, the internal passage of the cutting tool is selectively connectable to a vacuum generator or a pressure reservoir via a valve, and the method comprises switching the valve from a first position in which the internal passage is in fluid communication with a vacuum generator to a second position in which the internal passage is in fluid communication with a pressure reservoir.

[0015] The pressure difference between the pressure reservoir and the sealed collection tube generates a pressure pulse that discharges the biological material from the filter element into the collection tube.

[0016] In a preferred embodiment, the method comprises a further step of using a vacuum generator to remove air from the collection tube at least partially before the second end of the tool is connected to a pressure reservoir. The advantage of evacuating the vacuum tube is that this increases the differential pressure between the sealed collection tube and the pressure reservoir. A further advantage of evacuating the collection tube is that the atmosphere may act as the pressure reservoir, and the step of creating overpressure may simply involve exposing the evacuated tube to atmospheric pressure.

[0017] Preferably, the pressure reservoir supplies a pressure higher than atmospheric pressure, for example, 200-400 kPa, to ensure that the generated pressure pulses are sufficient to discharge biological material that is inherently sticky and may adhere to the underside of the filter element. The sealing contact between the collection tube and the connection interface in the cutting tool is accompanied by a predetermined clamping force. Preferably, the pressure reservoir supplies a pressure higher than the pressure in the sealed collection tube and lower than the clamping force between the collection tube and the tool.

[0018] In embodiments of a method in which the step of generating overpressure includes generating a pressure higher than atmospheric pressure, the method appropriately comprises a subsequent step of equilibriuming the collection tube with atmospheric pressure. This can be achieved via controlled leakage of the pressure reservoir, by switching a valve to a third position in which the internal passage is in fluid communication with the atmosphere, or by other suitable means. The collection tube can then be removed from the cutting tool.

[0019] Appropriately, the method comprises controlling the airflow during the cutting step. In a further development, the method comprises the step of measuring the pressure or airflow downstream of the filter element of the cutting tool, or the step of estimating the area of ​​biological material cut from a glass slide. The airflow is controlled by adjusting the aperture of a variable restrictor in response to the measured pressure or measured airflow, or the estimated area of ​​the cut material. The advantages of the further development will be described later with respect to the apparatus of the invention.

[0020] In a second aspect, the present invention defines a cutting tool for use in a method that is disposable and can be manufactured simply and at low cost. The tool has a first and a second end, an internal passage extending through the body of the tool, and a breathable filter element disposed in the internal passage. An orifice and a scraping blade are provided at the first end of the tool. The scraping blade is preferably made of a metal having a high yield stress. The tool body is preferably made of a polymeric material. At the second end of the tool, a connection interface is provided for releasably engaging the cutting tool in an airtight manner with a corresponding interface of a tool carrier.

[0021] According to the present invention, the tool body comprises a monolithic body portion, i.e., is formed as one piece. The filter element is fixedly held within the body portion, and the mechanical interface comprises one or more conical recesses or one or more conical protrusions formed in the body portion.

[0022] The tool is intended to be discarded after use. Thus, it is advantageous for the tool to be as inexpensive as possible, which is achieved, at least in part, through a simple manufacturing process. The method of the present invention allows the cut material to be removed from the interior of the tool without the need to disassemble the tool. Thus, the body portion holding the filter element may incorporate additional functions of the mechanical interface and may be implemented as a single piece, thereby minimizing the number of different parts that need to be assembled to manufacture the tool.

[0023] In one embodiment, the main tool body portion incorporates a scraping blade, and the entire tool body is a monolithic part. In a preferred embodiment that can enhance manufacturing precision, the scraping blade is provided on a second "blade" portion joined to the body portion. Suitably, the blade portion is irreversibly joined to the body portion, i.e., the two portions cannot be separated without breaking the bond. This can be achieved via fitting, adhesive bonding, or other suitable joining methods. The irreversible bond enhances the robustness of the tool body as a whole. Thus, the cutting tool according to the present invention may comprise at most two body portions.

[0024] Preferably, the main tool body portion and the blade portion, if present, are formed in a molding process from a suitable polymeric material. A metallic scraping blade may be embedded on one side of the tool orifice or may be attached via adhesive bonding or other suitable joining methods. In other embodiments, the blade portion or the entire tool body is overmolded with the scraping blade. The molded material can be an injection molding grade thermoplastic polymer. The grade can be a glass-filled, ceramic-filled, or carbon-filled material. The polymer can be from polypropylene, polyester, polycarbonate, ABS, or other engineering thermoplastic materials and mixtures thereof commonly used in engineering and high-precision applications.

[0025] The connection interface for attaching the tool to the tool carrier of the cutting device is integrally formed with the monolithic main tool body portion. For high-precision cutting, it is important that the tool be attached with accurate alignment, which is achieved via a conical interconnection. In one embodiment, the upper end portion of the main tool body portion comprises a plurality of conical pins that engage with corresponding conical recesses provided in the tool carrier. Alternatively, the tool may comprise a plurality of conical grooves configured to engage with corresponding conical pins in the tool carrier.

[0026] In a preferred embodiment, the tool body portion includes a single conical groove that forms part of the internal passage.

[0027] In addition, the second end of the tool may be provided with a click fitting or snap-fit ​​element for establishing a quick coupling with a tool carrier. The quick coupling element is preferably also formed on the body portion of the cutting tool. The advantage of the quick coupling is that it facilitates automated attachment and detachment of the tool after cutting and transport.

[0028] The tool further comprises a second connection interface for establishing a seal with the collection tube when positioned around the tool orifice. In one embodiment, the second connection interface comprises a collar having an outer diameter sized to match the inner diameter of the collection tube. The collar may have a larger diameter flange portion, the lower side of which is used to contact the upper rim of the collection tube when positioned around the orifice. A seal may then be formed between the upper rim and the lower side, thereby pressing the collection tube against the flange portion with a predetermined clamping force to achieve an airtight connection. Optionally, the lower side of the flange portion may be provided with a layer of relatively flexible material to enhance the seal. The collar may have a conical outer surface that engages airtightly with the inner rim of the collection tube.

[0029] Advantageously, to facilitate manufacturing, the second connection interface is formed integrally with the tool body. The second connection interface may also be formed by a separate collar made of an elastic material that is mounted around the tool body. Examples of suitable materials include rubber (crosslinked rubber, olefin, urethane, or silicone) and thermoplastic elastomers, such as polyester, polyurethane, and polyolefin. Part of the collar may have an outer diameter slightly larger than the inner diameter of a standard-sized collection tube.

[0030] The tool of the present invention further comprises a permeable filter element positioned within the internal passage and extending across its entire diameter. Preferably, the filter element is made of a porous material having pore sizes that do not allow the passage of the material being cut, e.g., 10, 50, 100, or 200 μm, depending on the sample being cut. The filter element may have a woven or unwoven structure and is preferably made of a synthetic material, e.g., polyethylene, polypropylene, polyvinyl chloride, polyester, nylon, polyvinylidene fluoride, or Teflon®. The filter element may have a diameter of 3.0 to 15.0 mm. Alternatively, the filter may be a thin substrate having a defined array of pores formed by lithography, etching, or laser ablation.

[0031] In a preferred embodiment, the filter element material is held in a substantially circular frame portion, which is a separate portion inserted into and fixed within the internal passage of the main tool body. The frame portion may be overmolded onto the filter element material. In one embodiment, the frame portion has a diameter larger than the diameter of the internal passage of the tool and is held radially via an interference fit.

[0032] In a further embodiment, the main tool body is overmolded onto the filter element.

[0033] The filter element divides the internal passage into first and second sections. The first section of the passage is defined as the section between the filter element and the tool orifice. The first section of the passage is preferably linear and has a longitudinal central axis L. The diameter of the first section of the passage may be constant over its length or may vary. For example, the passage may narrow toward the tool orifice. When in use, the portion of the tool body surrounding the first section of the passage functions as a suction nozzle and is called the tool nozzle.

[0034] The scraping blade is positioned on one side of the tool orifice. The scraping blade has a scraping edge of width w that contacts the biological material on the slide. In some embodiments, the blade is a flat, chisel-like member. In other embodiments, the scraping blade is curved about the blade axis and includes a thin-walled tube section. The tube section may be a continuous thin-walled cylinder or a partial cylinder such as a semi-cylinder. The end faces of the tube section function as scraping edges.

[0035] In some embodiments of a cutting tool that may be used in an apparatus according to the present invention for carrying out the method of the present invention, the nozzle is partially formed from a thin-walled tube section, and the scraping blade forms part of the nozzle. The tube section may have a wall thickness of 30 to 100 μm and an outer diameter of 3.0 to 15 mm. The scraping edge of the tube section or the scraping edge of a flat blade may be perpendicular to the blade axis or angled for optimized scraping performance. In some embodiments, the blade has a polished sharp edge.

[0036] Typically, the cutting tool used in the present invention is positioned such that the scraping blade engages with the horizontal plane of the glass slide at a predetermined angle α, where this angle is defined as the scraping angle. In some embodiments, the scraping blade extends parallel to the longitudinal axis L of the nozzle, and the scraping angle is defined by the orientation of the nozzle axis relative to the slide surface.

[0037] In other embodiments, particularly when the scraping blade extends from the end face of the nozzle, the scraping blade has a blade axis that extends at a predetermined angle with respect to the longitudinal axis L. Thus, the nozzle may be positioned such that the longitudinal axis L is perpendicular to the slide surface, and the scraping angle is defined by the angle between the slide surface and the blade axis. As can be understood, the scraping angle α can be varied by adjusting the angular orientation of the nozzle axis.

[0038] In further developments, the scraping blade of the cutting tool is provided with an anti-sticking coating such as NiF or perfluoroalkane to prevent the cut material from sticking and to ensure that the cut material is adsorbed to the underside of the filter element.

[0039] In a further development, the nozzle includes a nozzle extension that at least partially surrounds the tool orifice on the side opposite to the scraping blade. In one embodiment, the tool is configured to be positioned at a predetermined scraping angle α with respect to a horizontal slide surface. When the blade contacts the slide, the periphery of the nozzle extension may be configured to form a certain gap with respect to the slide surface. In a further embodiment, the periphery of the nozzle extension has a predetermined contour such that the gap changes along the circumferential direction.

[0040] The advantage of the nozzle extension is that it optimizes the airflow generated during suction at the location of the scraping edge, thereby ensuring that all cut material is drawn into the tool body.

[0041] Therefore, the cutting tool used in the method of the present invention performs three functions: cutting, collecting, and transporting. Furthermore, since the tool is intended to be discarded after use, the risk of secondary contamination of biological material is further reduced.

[0042] In a third embodiment, the present invention relates to an apparatus for cutting and transferring biological material from a sample placed on a flat substrate, configured to perform the above-described embodiment of the method of the present invention. The apparatus is A tool carrier having a mechanical interface, wherein the mechanical interface comprises an internal passage extending between a first end and a second end of a tool, a filter element positioned inside the cutting tool so as to extend across the internal passage, and a scraping blade positioned in the orifice of the tool at the first end of the tool, to establish an airtight connection with the corresponding mechanical interface of a cutting tool. • A platform for supporting a flat substrate. • A stage for holding at least one collection tube. A positioning system configured to move a tool carrier and a platform relative to each other and to control their relative positions so that the scraping blade of a cutting tool selectively engages with biological material in a given area of ​​the sample, and further configured to move the tool carrier relative to the stage so that the collection tube is positioned around the tool orifice. • Vacuum generator or mounting fixture for connecting an external vacuum generator, • Pressure reservoir or fitting for connecting an external pressure reservoir, and A valve is provided to discharge the collected biological material into the collection tube, which is operable between a first position where the internal passage of the cutting tool communicates with a vacuum generator and a second position where the passage communicates with a pressure reservoir. It is equipped with.

[0043] In one embodiment, the pressure reservoir is simply atmospheric pressure. In a further embodiment, the pressure reservoir is a pump. In a further embodiment, the pressure reservoir is a container that holds superpressurized air held at a pressure of 100 to 400 kPa.

[0044] Preferably, the apparatus includes an image acquisition system for acquiring images of a biological sample. Appropriately, the image acquisition system is configured to identify the boundary between a region of interest containing the biological material to be tested and an undesirable region containing material not to be tested. In other words, the image acquisition system identifies the shape of the region to be cut. In one embodiment, the biological sample is a colored tissue sample, and the image acquisition system simply recognizes the colored region. In other embodiments, the image acquisition system may be programmed with software to process the captured images of the tissue sample and identify the region of interest, for example, based on cellular structure. The system may be configured to identify the shape of the region to be cut by comparing the captured images with reference images marked by a pathologist. Appropriately, the image acquisition system is configured to transfer the coordinates of the identified boundary to a control device of a positioning system.

[0045] The scraping blade of the cutting tool has a scraping edge of width w. As a result of the relative movement of the blade and the flat substrate in the translational direction, the scraping edge cuts a track through the biological sample of the corresponding width. If the blade is formed by a section of a thin-walled tube, the width of the track depends on the outer diameter of the tube and the applied contact force.

[0046] During cutting, the cutting tool may be positioned so that the scraping blade is at a scraping angle of 30 to 60 degrees, although other angles may be preferable depending on the properties of the biological sample being cut.

[0047] In further developments, the tool carrier is pivotally mounted to the device, thereby allowing adjustment of the scraping angle and, if necessary, bringing the cutting tool to a vertical position, facilitating, for example, automated coupling / uncoupling of the cutting tool around the tool's sealing collar and / or automated mounting of the collection tube.

[0048] Appropriately, the positioning system includes electric actuators for relative movement in the lateral X and Y directions and the vertical Z direction. In one embodiment, the positioning system further includes a rotating stage for adjusting the position of the platform (and biological sample) relative to the scraping blade about a rotation axis perpendicular to the flat substrate.

[0049] During cutting, the cutting tool is moved relative to the platform in the translational direction. In a further development, the tool carrier of the device is rotatable about an axis perpendicular to the platform to allow for angle adjustment of the scraping edge relative to the translational direction. This is advantageous because it allows for variation of the effective width of the scraping edge, i.e., the width measured perpendicular to the translational direction. Appropriately, the positioning system is equipped with actuators for controllable adjustment of the angular orientation.

[0050] The actuators of the positioning system may be coupled to a platform to move a biological sample relative to a cutting tool, and / or the actuators may be coupled to a tool carrier to move the cutting tool relative to the platform.

[0051] In further developments, the device is equipped with an ion generator. Contact between the scraping edge and the flat surface on which the biomaterial is placed generates friction, which can lead to the accumulation of static charge in the biomaterial. The ion generator regulates the airflow around the cutting tool, thereby reducing the accumulation of static charge that could negatively affect the collection of biomaterial during cutting.

[0052] In further developments, the device includes a variable restrictor positioned side-by-side between the vacuum generator and the filter element of the cutting tool to regulate the airflow through the tool nozzle. When the tool is in use, the cut material collected below the filter element restricts the airflow through the filter, thereby increasing the overall restriction of the system. This results in pressure loss and reduced airflow, which can adversely affect the suction capacity of the tool nozzle. Appropriately, the device is configured to reduce the restriction of the variable restrictor in response to the accumulation of cut material below the filter, in order to allow a constant airflow through the internal passages and nozzle.

[0053] In one embodiment, the apparatus further includes a pressure sensor or flow sensor positioned side-by-side between the vacuum generator and the filter element, and the variable restrictor is controlled based on the measured pressure or measured flow rate. In a further embodiment, the apparatus is configured to estimate the area of ​​biological material cut by the scraping blade and to reduce the limit of the variable restrictor with respect to the cut area. The image acquisition system may be configured to estimate the cut area, or the cut area may be calculated based on the width of the scraping blade and the distance of each translational movement of the blade relative to the slide.

[0054] Those skilled in the art will recognize that two or more of the above-described embodiments, practices, and / or aspects of the invention may be combined in any way deemed useful.

[0055] The invention will now be further described with reference to the embodiments described below. [Brief explanation of the drawing]

[0056] [Figure 1a] This figure schematically shows an example of an apparatus according to the present invention, configured to carry out the method of the present invention for cutting biological material from a glass slide and transferring it to a matching collection tube. [Figure 1b]This figure shows a portion of the apparatus in Figure 1a, in a configuration where the material is removed from the slide and drawn into the cutting tool. [Figure 1c] This figure shows a portion of the apparatus in Figure 1a in a configuration prepared for the transfer of the aspirated material to the collection tube. [Figure 1d] This diagram shows a portion of the apparatus in Figure 1a, in a configuration where the aspirated material is transferred to a collection tube. [Figure 2] This is a side view of an example of a cutting tool for use in the apparatus and method according to the present invention. [Figure 3] This is a side view of an example of a cutting tool according to the present invention, with the main internal features indicated by dotted lines. [Figure 4a] This is a cross-sectional side view of a further embodiment of the cutting tool according to the present invention. [Figure 4b] Figure 4a is a cross-sectional view of the filter element attached to the tool.

[0057] It should be noted that items with the same reference number in different drawings have the same structural features and functions or signals. While the function and / or structure of such items has been described, repetition of that description is unnecessary in the detailed description. [Modes for carrying out the invention]

[0058] Pathological diagnostic studies of biological materials such as tissues and cells form the basis for many treatment decisions, particularly in oncology. For example, genome-based studies are performed to inform treatment options for individual patients diagnosed with cancer. Biological material / tissue may be obtained from a biopsy and then cut into thin slices, which are, for example, embedded in paraffin and fixed on a glass slide. These thin slices are called tissue samples. Other methods for obtaining and preparing biological material are known.

[0059] A tissue sample has a region of interest (ROI) containing the material to be tested for diagnosis. The ROI can be identified by coloring, or the pathologist may provide a marking on a reference slide after microscopic analysis. The ROI can also be identified through processing of a digital image of the sample. Once the ROI is identified, the material is removed / cut from the slide and then transferred to the analyzer. Typically, the material is transferred to a collection tube, where sample preparation process steps such as cell lysis, purification, and amplification, and any further necessary processing steps, are performed. As is understood, the reliability and efficiency of the analysis are optimized by ensuring that only material cut from the ROI is present, but also by minimizing contamination.

[0060] The present invention defines an automated method for cutting biological material from a sample placed on a flat substrate, enabling precise material removal, and transferring the biological material to a collection tube while minimizing contamination of the material to be analyzed. The method uses a cutting tool that can be selectively connected to a negative pressure source and an overpressure source. The cutting tool and apparatus for carrying out the method of the invention are described with reference to Figure 1a.

[0061] Figure 1a schematically shows an example of an apparatus according to the present invention. The apparatus 100 comprises a platform 110 for supporting a slide glass 115 on which a biological sample 117 is placed, and a cutting tool 120 mounted on a tool carrier 130. The tool carrier is mounted on a positioning unit 135 which allows the position of the tool 120 to be adjusted relative to the platform 110. In the example shown, the positioning unit 136 moves the tool 120 vertically in the z direction and rotates it about a vertical axis. The positioning unit forms part of a positioning system which allows the movement of the tool relative to the platform in the translational directions X and Y. Preferably, the platform 110 is also movable, and the positioning system comprises a control device 190 which controls the relative movement so that the tool engages with the biological material 117 only in a region of interest. In the example shown, the apparatus further comprises an image acquisition system 180 for locating tissue and providing ROI coordinates to the control device 190.

[0062] The cutting tool has a substantially hollow body that defines an internal passage extending between the first and second ends of the tool body. A breathable filter element 125 is housed within the tool body and extends across the entire diameter of the internal passage. The first section of the internal passage is defined between the filter element 125 and the tool orifice 123 at the first end of the tool. The portion of the tool body surrounding the first section of the internal passage is defined as a nozzle 122 having a longitudinal central axis L. The nozzle axis L may, but is not required, coincide with the central axis of the entire tool, as in the example in Figure 1a. The second end of the cutting tool has a mechanical interface for releasably connecting the tool to a corresponding mechanical interface on the tool carrier 130.

[0063] In the example shown, the nozzle is formed by a thin-walled tube, and a portion of the end face of the tube functions as a scraping blade 124 (see Figure 1b) used to mechanically remove the biological material 117 placed on the glass slide 115. The scraping blade may be a separate portion extending from the nozzle end face on one side of the orifice 123.

[0064] During cutting, the tool is positioned so that the nozzle 122 is directed at an angle α with respect to the horizontal plane of the platform 110, and angle α also defines the scraping angle of the blade 124. The tool carrier 130 is rotatably mounted on the positioning unit 135 to allow adjustment of angle α.

[0065] The apparatus further comprises a vacuum pump 150, a pressure reservoir 160 having a pressure of at least 100 kPa, a valve 140, and appropriate tubing arranged such that the internal passage 121 of the cutting tool 120 can be selectively connected to either the pump 150 or the pressure reservoir 160. In the example shown, the valve is a three-way valve, with a first port 141 in fluid communication with the mechanical interface and passage 121 in the tool carrier 130, a second port 142 in fluid communication with the vacuum pump 150, and a third port 143 in fluid communication with the pressure reservoir 160.

[0066] Here, embodiments of the method of the invention will be described with reference to Figures 1b to 1d, but for simplicity, only the relevant features of the apparatus are shown.

[0067] Figure 1b shows the cutting tool at the material removal position, in which, in the first step of the method, the biological material 117 is scraped off the glass slide 115 and collected in the tool body. The tool 120 is positioned so that the scraping blade 124 engages with the slide surface at a scraping angle of, for example, 30 to 60 degrees. The tool is then moved relative to the slide in the translational direction x, thereby causing the scraping edge of the blade, having a predetermined width in the y direction, to cut the track through the biological material 117. The relative position of the scraping blade 124 and the slide 115 is appropriately controlled so that only material from the identified ROI is cut through the scraping.

[0068] Simultaneously with the scraping step, a negative pressure is formed at the second end of the cutting tool, and this negative pressure creates an upward airflow at the tool orifice 123, drawing the cut material into the passage 121, where the material is captured below the filter element 125. During this suction step, the valve 140 is in a first position, with the first and second ports 141,142 in fluid communication and the internal passage 121 in communication with the vacuum pump 150. The airflow holds the cut material below the filter element 125 during the material removal process.

[0069] Once cutting is complete, the tool position may be adjusted to a transfer position where the platform and the cutting tool are separated from each other. In the next step of the method, the collection tube 170 is airtightly attached to the outer surface of the cutting tool 120 as shown in Figure 1c. Appropriately, the apparatus includes, for example, a stage for holding the tray of the collection tube, and a positioning system is configured to control the movement of the tool carrier relative to the stage so that the tool 120 is inserted into the tube 170. The valve 140 remains in the first position, and the cut material 117 is held below the filter element 125. As a result of continuous connection with an operating vacuum pump 150, air is discharged from the collection tube 170.

[0070] The final step of the method involves exposing the filter element 125 to overpressure at the second end of the tool 120. This is achieved by switching the valve 140 to a second position in which the first port 141 is in fluid communication with the third port 143 and the pressure reservoir 160, as shown in Figure 1d. Preferably, the valve is switched to the second position when a pressure approximately equal to a vacuum or the absolute pressure of a vacuum pump, e.g., 1 to 10 kPa, is achieved inside the tube 170. When exposed to overpressure, a pressure pulse and airflow are generated that flows toward the tool orifice 123, as indicated by the arrow in Figure 1d. The aforementioned pressure pulse pushes the cut material 117 from the underside of the filter element 125 into the collection tube 170.

[0071] The pressure reservoir 160 can simply be atmospheric pressure. Preferably, a reservoir of pressurized air held in a pump or cylinder is used to supply a pressure of 200-400 kPa to generate pulses of sufficient magnitude to ensure that all material is discharged from the filter element 125. Preferably, the supplied pressure is lower than the clamping force produced by the airtight connection of the collection tube 170 around the outer surface of the tool body. This prevents the tube from being released by the pressure pulse, which would create a risk of loss of cut material.

[0072] After being exposed to a pressure higher than 100 kPa, the pressure inside the collection tube is brought to equilibrium with the atmosphere. The collection tube may then be removed from the cutting tool 120 and properly covered with an end cap.

[0073] Therefore, the method of the present invention enables automated cutting and transfer of biological material from a sample placed on a glass slide 115 into a collection tube 170 without the need to use any liquid during cutting or to "rinse" the cut material from the tool body. This not only simplifies the process but also minimizes contamination.

[0074] Figure 2 shows an example of a cutting tool 220 suitable for use in the apparatus according to the present invention. The tool has a hollow tool body having an internal passage in which a filter element is arranged. A nozzle 222 is provided at the first end 220a of the tool, and a portion of the nozzle functions as a scraping blade 224 having a scraping edge 224a that is brought into contact with the slide glass 115 and moved relative to the slide to cut the material. In this example, the scraping blade 224 is a separate component formed from a thin-walled tubular section made of a metal having a high yield strength. The rest of the nozzle 222 and the tool body may be formed from a polymer material overmolded onto the scraping blade 224. Preferably, the inner surface of the scraping blade is provided with an anti-sticking coating to prevent the cut material from sticking during scraping and transfer.

[0075] When the tool is in use, a portion of the end face of the nozzle 222 that contacts the biological material on the slide 115 functions as a scraping blade 224. Thus, the scraping edge 224a in the shown example is arc-shaped and has a predetermined width to cut the track through the biological material when the underside of the nozzle is in contact with the slide surface and moved relative to the slide. Thus, the cut material may be ribbon-shaped.

[0076] As explained, the negative pressure generated by the vacuum pump creates an upward airflow that draws in the material cut by the scraping edge 224a. To ensure that the ribbon of cut material is drawn into the tool body, it is important that the buoyancy effect of the airflow is optimized at the location of the scraping edge. In the example shown, this is facilitated by an extension 226 of the nozzle 222 that surrounds approximately half of the tool orifice on the circumferential side opposite the scraping edge 224a. The extension 226 forms a curved hood, so that the maximum length of the extension 226 measured in the longitudinal direction L relative to the scraping edge 224a occurs at the circumferential position of the nozzle at 180 degrees relative to the scraping edge. On each side of this 180° position, the length of the nozzle extension is reduced.

[0077] In the example shown, the cutting tool 220 is configured to be mounted on a tool carrier such that the scraping blade 224 is positioned at a scraping angle of 45 degrees with respect to the horizontal plane of the slide. The blade axis of the scraping blade extends parallel to the central axis L of the nozzle and the tool, which means that the scraping angle coincides with the angle of the tool axis with respect to the surface of the slide.

[0078] The scraping edge 224a forms part of the suction nozzle. The peripheral edge of the extension 226 also forms part of the nozzle, thereby shortening the gap G between the nozzle and the slide surface, and thereby increasing the suction force applied to the cut biological material. The extension 226 is designed so that the gap G is substantially constant when the tool is positioned at a 45-degree angle to the slide surface and the scraping edge is in contact with the slide. In other examples, the nozzle extension is designed so that the gap changes along the circumference.

[0079] In addition to enhancing the levitation effect at the scraping edge 224a, the extension 226 can also function as a physical barrier to prevent the cut ribbon of the material from deviating laterally and to hold it within the nozzle.

[0080] At the second end 220b of the tool, a mechanical interface is provided for releasably mounting the tool 220 to a corresponding interface on the tool carrier. The interface appropriately allows for an airtight connection, ensuring optimal suction and advanced mechanical alignment through the tool body, thereby facilitating the precise positioning of the scraping edge 224a relative to the slide 115. For speed and automation purposes, click-type, clamping, or bayonet mountings are advantageous, but other types of interfaces, such as threaded connections, are also possible.

[0081] The cutting tool 220 further includes an external connection interface for receiving a collection tube positioned on the tool body. In the example shown, the outside of the tool body is fitted with a collar 227 made of elastomer sealing material to establish an airtight connection with the collection tube. Thus, the dimensions of the collar depend on the collection tube used. Preferably, the collar has a chamfered edge 227a for guiding the open end of the collection tube onto the main portion 227b of the collar, and the main portion 227b has an outer diameter that seals against the inner diameter or rim of the collection tube. Appropriately, the collar also has a larger diameter flange portion 227c that holds the rim of the collection tube to prevent damage that may otherwise occur when the tube is fitted and vented.

[0082] Figure 3 shows a side view of an example of a cutting tool according to the present invention. The tool 320 comprises a first main body portion 326 formed as a single piece. An internal passage 321 extends through this body portion 326, and a filter element 325 (shown by a dotted line) is positioned in the passage 321 and held in the main body portion. A mechanical interface for releasably connecting the tool to a carrier is provided in the main body portion 326 at the second end 320b of the tool. The mechanical interface comprises a plurality of conical recesses surrounding the internal passage 321 at the second end 320b of the tool.

[0083] The tool further comprises a second section 327 incorporating a nozzle section 322. The second section may be formed from a polymer material overmolded onto a main body section 326. The scraping blade 324 is embedded in the end face of the nozzle section 322. It is also possible that the nozzle section and the main body section 326 as a whole are formed from a material overmolded onto a metal scraping blade 324.

[0084] In the example shown, the second part 327 not only comprises the nozzle 322 and the blade 324, but also further comprises a second mechanical interface or collar for connecting the collection tube. As shown in the example in Figure 2, the collar has a flange portion 327c for holding the rim of the collection tube.

[0085] In this example, the tool 320 is configured to be positioned such that the longitudinal central axis L of the nozzle is perpendicular to the glass slide 115 during cutting. At the first end 320a of the tool, the end face of the nozzle 322 surrounds the orifice 323 at the entrance to the internal passage 321, indicated by a dotted line. A metal scraping blade 324 is embedded in the nozzle, with a portion of the blade protruding from the end face of the nozzle. The embedded portion of the blade is indicated by a dotted line. In the example shown, the scraping blade is formed from a thin piece of metal bent into a semi-cylindrical shape about the blade axis B. The scraping blade may also be a straight, flat piece similar to a chisel.

[0086] The blade axis B extends at a predetermined angle with respect to the nozzle axis L, and the scraping angle is defined by the angle between the blade axis B and the slide surface. The scraping angle can, of course, be changed by adjusting the orientation of the tool body. Appropriately, the scraping blade 324 extends toward the nozzle axis L such that the scraping edge 324a of the blade is positioned below the tool orifice 323.

[0087] Further examples of tools according to the present invention are shown in a cross-sectional side view in Figure 4a. The tool 420 comprises a main body portion 426 formed from a single piece through which an internal passage for the tool extends. The diameter of a portion of the internal passage 421 tapers toward a tool orifice 423. Further portions of the internal passage are formed by a conical recess 450, which is configured to connect the tool in precise alignment to a correspondingly shaped conical projection on the tool carrier. As understood, the conical projection has an internal passage that connects with the internal passage for the tool. The conical recess 450 provides an airtight connection with the tool carrier projection. In some examples, the coupling force between the tool and the tool carrier is established via an interference fit. In the example shown, the first connection interface additionally comprises a quick-release coupling in the form of a snap-fit ​​joint. Multiple snap-fit ​​joints 455, three in the shown embodiment, are provided at the entrance to the conical recess and are configured to engage with annular ridges provided on the conical projection of the tool carrier when the conical projection of the tool carrier is pressed into the tool recess 450. In the shown example, the snap-fit ​​joint is a cantilever arm extending into the corresponding recess 457 of the tool body 426 at the entrance to the conical recess 450. The gap between each arm 455 and the radially outer wall of the recess 457 allows for the deflection of the arm required for engagement and disengagement of the tool. Appropriately, the snap-fit ​​joints 455 are arranged at equal angular intervals around the entrance to the conical recess 450 and are configured to provide a stable locking force that is large enough to allow precise cutting with the tool, but small enough to allow disengagement with relatively low force.

[0088] The main tool body portion 426 further includes a seating portion 430 for positioning a filter element 425 in the axial direction. An example of a suitable filter element is shown in the cross-sectional view of Figure 4b. The filter element comprises, for example, a thin piece of porous, breathable woven material having a thickness t of 0.1 mm. Typically, a membrane filter having a larger thickness, for example, 0.6 mm, may be used. The filter element 425 is held in a frame portion 425a which can be molded around the outer circumference of the filter element. The outer diameter of the frame portion 425a is slightly larger than the diameter of the internal passage at the position of the seating portion 430 for the filter element 425, thereby holding the frame portion 425a radially within the main body portion 426 by an interference fit.

[0089] The scraping blade 424 of the tool is provided on a second portion 427 which is joined to the main body portion 426. The second portion 427 may be overmolded onto the scraping blade 424 and irreversibly joined to the main body portion 426 by a geometric fit, adhesive bonding or other suitable bonding method. The second portion 427 also includes a tool orifice 423 and part of the nozzle 422. The outer surface of the nozzle portion 422 in the second body portion 427 is configured to be inserted into the collection tube. The tool body further has a relatively flat lower surface 428 for receiving the rim of the collection tube and establishing a sealing connection with the rim of the collection tube. This sealing surface 428 may be provided on one or both of the main body portion 426 and the second portion 427. Alternatively, the sealing surface may be formed by the conical outer surface of the nozzle portion 422.

[0090] After cutting and tissue transfer, the cutting tool according to the present invention is discarded, and a new tool is connected to the device, thereby eliminating the risk of secondary contamination.

[0091] Whether indicated as non-limiting or not, examples, embodiments, or optional features should not be understood as limiting the invention described in the claims. It should be noted that the embodiments described above are illustrative, not limiting, and that those skilled in the art can design many alternative embodiments without departing from the scope of the appended claims.

[0092] In a claim, any reference numerals placed between parentheses should not be construed as limiting the claim. The use of the verb “to equip” and its conjugations does not preclude the existence of elements or steps other than those described in the claim. The article “a” or “an” preceding an element does not preclude the existence of multiple such elements. The present invention may be carried out by hardware comprising multiple distinct elements and by a appropriately programmed computer. In an apparatus claim listing multiple means, multiple of these means may be embodied by one identical item of hardware. The mere fact that a means is referenced in different dependent claims does not imply that combinations of these means cannot be used advantageously. [Explanation of Symbols]

[0093] 100 cutting equipment 110 platforms 115 slides 117 Tissue samples 120, 220, 320, 420 Cutting Tools 121,321,421 Internal passages through the cutting tools 122,222,322,422 nozzles 123,323,423 Tool Orifice 124,224,324,424 Cutting tool scraping blades 125,325,425 breathable filter elements 425a Frame portion of the filter element 130 Tool Carrier 135 Positioning Unit 140 3-way valve 141, 142, 143 First, second, and third valve ports 150 Vacuum pump 160 Pressure Reservoir 170 collection tubes 180 Image Acquisition System 190 Control device for positioning system 220a, 320a First end of cutting tool 220b, 320b Second end of the cutting tool 224a,324a Scraping edge of scraping blade 226 Nozzle extension 227,327 sealed colors 227a Chamfered edge of sealing collar 227b Body in sealed color 227c, 327c sealing collar retaining flange 326,426 First main tool body 327,427 The second part into which the blade is incorporated 428 Sealing surface of the tool body 430 The seating area in the main tool body for positioning filter elements 450 Conical recess for establishing a tool carrier and airtight interface 455 Snap-fit ​​joint 457 Recess of the tool body at the entrance to the conical recess L Tool nozzle longitudinal axis B Blade axis α: Blade scraping angle relative to the slide surface G Gap between nozzle and slide surface

Claims

1. A cutting tool (320, 420) for removing and collecting biological material (117) from a sample placed on a flat substrate (115), comprising a tool body through which an internal passage (321) extends between first and second ends (320a, 320b), the tool is A scraping blade (324, 424) is positioned in an orifice (123, 323) of the tool, which is provided at the first end (320a) of the tool, A first connection interface is provided on the second end (320b) of the tool to connect the cutting tool to an engaging interface in the tool carrier (130) in an airtight and releasable manner, A breathable filter element (325, 425) is positioned within the internal passage (321) so as to extend the entire diameter of the internal passage (321), Furthermore, The tool body comprises a main body portion (326, 426) formed as a single piece, and a first connection interface is integrally formed on the main body portion, thereby comprising at least one conical recess (450) or at least one conical projection. The filter elements (325, 425) are fixed and held within the main body portion (326, 426). The first section of the internal passage extending between the tool orifice and the filter element has a longitudinal central axis L, The scraping blade is positioned on one side of the tool orifice and has a semi-cylindrical tubular section bent about the blade axis B, so that the blade axis B extends obliquely toward the longitudinal central axis L of the first section of the internal passage. Cutting tool.

2. The cutting tool according to claim 1, wherein the scraping blades (324, 424) are incorporated into the main tool body portion (326, 426).

3. The cutting tool according to claim 1, wherein the scraping blades (324, 424) are incorporated into a second part (327, 427) joined to the main tool body part (326, 426).

4. The cutting tool according to any one of claims 1 to 3, wherein the filter elements (325, 425) are held in a separate frame portion (425a) fixedly held within the main tool body portion (326, 426).

5. The cutting tool according to any one of claims 1 to 3, wherein the main tool body portion (326, 426) is overmolded onto the filter element (325, 425).

6. The cutting tool according to any one of claims 1 to 5, wherein the first connection interface comprises a single conical recess (450) that forms part of the internal passage of the tool.

7. A cutting tool according to any one of claims 1 to 6, further comprising a second connection interface (327, 428) for establishing an airtight connection with a collection tube (170).

8. The cutting tool according to claim 7, wherein the second connection interface is integrally formed with the tool body and comprises a relatively large diameter portion (327c) having a lower portion (428) configured to receive the upper rim of the collection tube (170).

9. An automated method for transferring biological material (117) from a sample placed on a flat substrate (115) into a collection tube (170) using a cutting tool (120, 220, 320, 420) according to any one of claims 1 to 8, wherein the method comprises: a) A step of physically separating biological material from a flat substrate (115) using scraping blades (124, 224, 324, 424), b) step a) a step of generating a negative pressure at the second end (220b, 320b) of the tool in order to generate an updraft at the tool orifice (123, 323, 423), wherein the updraft draws the separated material into the internal passage (121, 321), where the separated material is held below the filter element (125, 325, 425), c) The step of positioning the collection tube (170) around the orifice so as to make sealing contact with the connection interfaces (227, 327, 428) in the cutting tool, d) A method comprising the steps of generating an overpressure at the second end of the tool to generate an airflow and pressure pulse to discharge the biological material held below the filter elements (125, 325, 425) and to transfer the material (117) into the collection tube (170).

10. The step of generating negative pressure comprises connecting the internal passages (121, 321) of the tool to a vacuum generator (150), The step of generating overpressure includes connecting the internal passage to a pressure reservoir (160), The method according to claim 9, further comprising the step of using a vacuum generator to remove air from a collection tube (170) before the internal passage is connected to a pressure reservoir.

11. The method according to claim 9 or 10, wherein step a) comprises bringing a scraping blade (124, 224, 324) into contact with a flat substrate (115) and moving a cutting tool relative to the flat substrate, and the method further comprises controlling the relative position of the cutting tool (120, 220, 320) and the substrate so that the scraping blade engages with the biological material only in the region of interest.

12. Step b) is The method according to any one of claims 9 to 11, comprising controlling the airflow through a cutting tool by changing the limit of a variable restrictor in response to pressure or airflow measured downstream from the filter elements (125, 325, 425) of the cutting tool.

13. The method according to any one of claims 9 to 12, wherein step d) comprises generating an overpressure higher than atmospheric pressure, and the method further comprises the step of equilibrating the collection tube (170) to atmospheric pressure before removing the collection tube from the cutting tool (120, 220, 320, 420).

14. An apparatus for cutting biological material from a sample placed on a flat substrate (115) and transferring it into a collection tube (170), configured to perform the method according to any one of claims 9 to 13, A tool carrier (130) having a mechanical interface for airtight connection with a cutting tool (120, 220, 320, 420) according to any one of claims 1 to 8, A platform (110) for supporting a flat substrate (115), A stage for holding at least one collection tube (170), A positioning system (135) is configured to move a tool carrier (130) and a platform (110) relative to each other and to control their relative positions so that the scraping blades (124, 224, 324) of the cutting tool selectively engage with the biological material (117) in a predetermined area, and further configured to move the tool carrier relative to the stage so that the collection tube is airtightly positioned around the tool orifice after cutting is complete, A mounting fixture for connecting a vacuum generator (150) or an external vacuum generator, A fitting for connecting a pressure reservoir (160) or an external pressure reservoir, To discharge the cut biological material into the collection tube, a valve (140) is provided that can operate between a first position in which the internal passages (121, 321) of the cutting tool communicate with a vacuum generator (150) and a second position in which the passages communicate with a pressure reservoir (160). A device equipped with the following features.

15. The apparatus according to claim 14, wherein the mechanical interface in the tool carrier (130) comprises a hollow conical projection for engaging with a corresponding conical recess (450) in the cutting tool.

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