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JP-7856252-B2 - Methods, tools, and apparatus for cutting and transferring biological materials.

JP7856252B2JP 7856252 B2JP7856252 B2JP 7856252B2JP-7856252-B2

Inventors

  • テイセン，エドビン・ヨハンネス・リシャルドゥス・ビルヘルムス
  • ハーゼン，ニコール・カタリーナ・エリーサベト
  • レンブレフツ，トーマス・パトリック・アンネ－リーセ
  • フィーマン，ケルシー
  • ビムベルガー－フリートル，ラインホルト

Assignees

  • キアル・ベー・フェー

Dates

Publication Date
20260511
Application Date
20210917
Priority Date
20200922

Claims (15)

  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 is: 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.

Description

This invention relates to an automated method for cutting biological material placed on a flat substrate, such as a glass slide, and transferring the cut material to a receptacle for further analysis. The invention further relates to related apparatus and disposable tools for use in the apparatus. For molecular analysis of a tumor to be performed for the purpose of tumor diagnosis, a certain amount and concentration of tumor cells must be present in the sample being analyzed. Tumor tissue is heterogeneous and contains other tissues and cell types. Therefore, 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 is the most common method, in which a laboratory assistant scrapes material from the ROI using, for example, a surgical scalpel blade, and transfers the scraped material into a collection tube. This requires a high level of skill, and even when performed by a highly skilled laboratory assistant, there is no guarantee that only material from the ROI will enter the collection tube. An improved manual method for tissue removal and collection is disclosed in U.S. Patent Application Publication No. 2020/038001. This method uses suction to remove tissue manually scraped from a slide. The cutting tool used has three main elements: a shaft portion, one end of which can be connected to a suction source; a cap incorporating a blade at the other end; and a filter column releasably fixed to the shaft portion via the cap. The filter column comprises a filter member that captures the cut material drawn 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 dissolving the cut tissue. 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 equipment. An example of an automated apparatus for extracting material from biological samples via milling is disclosed in U.S. Patent No. 1,0876,933. This 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. 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. U.S. Patent Application Publication No. 2020/038001U.S. Patent No. 10876933U.S. Patent No. 8,573,073 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.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.This figure shows a portion of the apparatus in Figure 1a in a configuration where the aspirated material is prepared for transfer to the collection tube.This diagram shows a portion of the apparatus in Figure 1a, in a configuration where the aspirated material is transferred to a collection tube.This is a side view of an example of a cutting tool for use in the apparatus and method according to the present invention.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.This is a cross-sectional side view of a further embodiment of the cutting tool according to the present invention.This is a cross-sectional view of the filter element attached to the tool shown in Figure 4a. It should be noted that items with the same reference n