Method for transferring tissue section

Abstract

This method for transferring a tissue section comprises: a swelling step for bringing a swelling agent into contact with a tissue section mounted on a primary plate; and a transfer step for transferring a portion of the tissue section to an analysis plate by bringing a transfer agent into contact with the tissue section, and then heating the tissue section in a state where the primary plate and the analysis plate are overlapped so that the tissue section comes into contact with the analysis plate. The swelling agent and the transfer agent each contain ammonium bicarbonate, acetonitrile, and water.

Description

Method for transferring tissue sections

[0001] The present invention relates to a method for transferring tissue sections.

[0002] Research institutions and hospitals often store large quantities of sections, such as paraffin-embedded sections and fresh-frozen sections, that have been observed and analyzed in the past. If these sections could be subjected to analysis, such as mass spectrometry imaging, new insights could be gained. However, if the original sections are processed for analysis, they will change, potentially making other analyses impossible or preventing the same observational analysis from being performed as before.

[0003] Therefore, there is a need for a technique to transfer a portion of the original section onto an analytical plate for mass spectrometry imaging or the like. For example, Non-Patent Document 1 discloses a technique in which a transfer plate created using proprietary glass formation and film deposition techniques is pressed onto the sample to transfer the data.

[0004] Hamamatsu Photonics K.K., "Transfer Plate Porepare™ A15551 Series", [online], June 2024, [Retrieved October 24, 2022], Internet <https: / / www.hamamatsu.com / content / dam / hamamatsu-photonics / sites / documents / 99_SALES_LIBRARY / etd / A15551_TPMZ1032J.pdf>

[0005] However, even if we were to attempt to transfer sections using the specially processed transfer plate described in Non-Patent Document 1, the specially processed transfer plate is more expensive than a general-purpose analytical plate, which would result in excessive costs.

[0006] This disclosure was made to solve the aforementioned problem, and its purpose is to transfer sections to a general-purpose analytical plate.

[0007] A method for transferring tissue sections according to the first aspect of this disclosure includes a swelling step of contacting a tissue section mounted on a primary plate with a swelling agent, and a transfer step of heating the tissue section with the primary plate and the analysis plate superimposed so that the tissue section is in contact with the analysis plate, after contacting the tissue section with a transfer agent, thereby transferring a portion of the tissue section to the analysis plate. The swelling agent and the transfer agent each contain ammonium bicarbonate, acetonitrile, and water.

[0008] According to the present invention, sections can be transferred to a general-purpose analytical plate.

[0009] This is a diagram illustrating the transfer method according to the embodiment. This is a diagram illustrating the transfer method according to the embodiment. This is a diagram showing the mass spectrum of the transfer. This is a diagram showing the mass spectrum of the original tissue section. This is a diagram showing the results of mass spectrometry imaging of the transfer and the original tissue section.

[0010] One embodiment of this disclosure (hereinafter referred to as "this embodiment") will be described below. However, this embodiment is not limited to this embodiment.

[0011] Furthermore, in this specification, "%" in a solution means "volume %" unless otherwise specified. This embodiment will be described in detail below with reference to the drawings. In the following, the same or corresponding parts in the drawings will be denoted by the same reference numerals, and their descriptions will not be repeated in principle.

[0012] [Embodiment] Figures 1 and 2 are diagrams illustrating the tissue section transfer method according to this embodiment. The dashed frame in Figure 1 shows the minimum configuration of the tissue section transfer method according to this embodiment. Figure 2 is a diagram showing the steps following the steps shown in Figure 1.

[0013] Referring to Figure 1, the method for transferring tissue sections according to this embodiment includes (i) a swelling step of contacting the tissue section with a swelling agent, and (ii) a transfer step of heating the tissue section while it is in contact with an analysis plate after the tissue section has been in contact with a transfer agent, thereby transferring a portion of the tissue section to the analysis plate.

[0014] Typically, the method for transferring tissue sections according to this embodiment includes (i) a swelling step of bringing a swelling agent into contact with the tissue sections mounted on a primary plate, and (ii) a transfer step of heating the tissue sections with the primary plate and the analysis plate superimposed so that the tissue sections come into contact with the analysis plate, thereby transferring a portion of the tissue sections to the analysis plate.

[0015] In this specification, "transcription" means transferring a portion of a tissue section to an analysis plate, as described above. The portion of the tissue section that has been transferred to the analysis plate (transcribed) is referred to as the "transcript." The two-dimensional distribution of a given component in the tissue section is carried over to the two-dimensional distribution in the transcript. More precisely, the two-dimensional distribution of a given component in the tissue section is inverted left to right compared to the two-dimensional distribution of the same given component in the transcript.

[0016] Furthermore, in this specification, “primary plate” means the plate on which the tissue sections were mounted before the transfer method according to this embodiment was performed. A primary plate is typically a plate used for observation, predetermined analysis, and / or preservation of tissue sections.

[0017] In one embodiment, the primary plate is a glass slide. In another embodiment, the primary plate is a plate for performing a predetermined analysis (for example, ITO glass, as described later).

[0018] Each of the swelling agent and transfer agent is ammonium bicarbonate (NH₃). ４ HCO ３ ) and acetonitrile (CH ３ Includes CN.

[0019] According to the tissue section transfer method of this embodiment, sections such as paraffin-embedded sections and fresh-frozen sections can be transferred to a general-purpose analytical plate. In this specification, an analytical plate is a plate used for a predetermined analysis (for example, mass spectrometry). Since an analytical plate is a plate for transferring and analyzing tissue sections from a primary plate, it can also be called a secondary plate.

[0020] (Name of Section) In this specification, unless otherwise specified, "tissue section" refers to a section that is ready for transfer by the swelling step and the transfer step described above, which constitute the minimum configuration of the tissue section transfer method according to this embodiment. A tissue section is, for example, a section that has been deparaffinized from a paraffin-embedded section, or a fresh frozen section. Typically, a tissue section is mounted on a primary plate such as a glass slide. A tissue section may also be a section that has not been preserved using paraffin, freezing technology, etc., after being cut from a living organism (hereinafter referred to as "untreated section" in this specification). In the following description, for convenience, sections taken during and after the transfer method according to this embodiment may also be referred to as "tissue sections".

[0021] Furthermore, in this specification, sections that have been preserved using methods such as paraffin or freezing are referred to as "preserved sections." Preserved sections are, for example, paraffin-embedded sections or fresh-frozen sections. Preserved sections are typically stored mounted on a primary plate.

[0022] Typically, untreated sections are mounted on a primary plate, and then preserved after or before observation or predetermined analysis of the tissue sections, becoming preserved sections. Preserved sections are stored mounted on the primary plate.

[0023] Furthermore, in this specification, "tissue sections" and "preserved sections" are collectively referred to as "sections." (Exploration of section transcription methods) Research institutions, hospitals, etc., often store a large number of preserved sections of specimens that have been observed and analyzed in the past, which have been preserved so that they can be observed and analyzed at a later date. Such preserved sections mainly include paraffin-embedded sections or fresh frozen sections.

[0024] The inventors investigated methods for using some of these preserved sections for analysis such as mass spectrometry. Because these preserved sections are extremely small in quantity and thin, slicing them parallel to a primary plate such as a glass slide is difficult. Furthermore, simply pressing a section from the primary plate onto another analysis plate did not transfer its components.

[0025] Therefore, the inventors conducted further intensive research and found conditions under which sections on a primary plate could be transferred to other analytical plates. Specifically, they found that by contacting the sections with an aqueous solution containing ammonium bicarbonate and acetonitrile, and then pressing the sections from the primary plate onto the analytical plate at a high temperature, the sections could be transferred.

[0026] Specifically, the inventors discovered that fresh frozen sections can be transferred to an analysis plate by the swelling and transfer steps described above. They also discovered that paraffin-embedded sections can be transferred to an analysis plate by performing the deparaffinization step described later, followed by the swelling and transfer steps described above.

[0027] The conditions of this embodiment, as discovered by the inventors, are described in detail below. (Swelling process) In the swelling process, the swelling agent is brought into contact with the tissue section. More specifically, in the swelling process, the swelling agent is brought into contact with the side of the tissue section mounted on the primary plate that is opposite to the primary plate (the side to be transferred, hereinafter also referred to as the "transfer surface"). In the following description, an example will be given in which the transfer surface of the tissue section is the upper surface of the tissue section, but the swelling process may also be performed with the transfer surface of the tissue section facing downwards. As a result of the swelling process, the swelling agent is contained in at least the vicinity of the upper surface of the tissue section. This makes it possible to change the state in which fibrous proteins (e.g., collagen fibers) and other components are tightly packed together in tissue sections that have undergone deparaffinization treatment after paraffin embedding, frozen fresh sections, etc., from a state in which there are gaps between them. In other words, highly hydrophobic tissue sections can be made hydrophilic and swelled.

[0028] The swelling agent contains ammonium bicarbonate, acetonitrile, and water. In one example, the concentration of ammonium bicarbonate in the swelling agent is 0.05 M or more and 0.15 M or less, and the concentration of acetonitrile in the swelling agent is 20% or more and 70% or less based on the swelling agent. The remaining component in the swelling agent is water (H ２ O) is correct. By configuring the swelling agent in this way, the subsequent transfer process can be carried out efficiently. The efficiency of the transfer process will also be referred to as "transfer efficiency" below.

[0029] In this specification, the first example of high transfer efficiency is that components contained in the tissue section are also detected in the transcript. The second example of high transfer efficiency is that the distribution of a predetermined substance in the tissue section is reflected in the transcript. Furthermore, the third example of high transfer efficiency is that the transfer process is simple.

[0030] The concentration of ammonium bicarbonate in the swelling agent is preferably 0.08 M to 0.12 M, and more preferably about 0.10 M. Ammonium bicarbonate is known to act as a buffer, maintaining the pH within a predetermined range.

[0031] The concentration of acetonitrile in the swelling agent is preferably 20% to 40%, and more preferably about 30%. By lowering the concentration of acetonitrile in the swelling agent compared to the concentration of acetonitrile in the transfer agent, the tissue sections can be swelled in the swelling process while suppressing the elution of components from the tissue sections more than in the transfer process.

[0032] Furthermore, in this specification, "contact" of an aqueous solution such as a swelling agent with a section may refer to either immersing at least a portion of the section in the aqueous solution contained in a container, or pouring the aqueous solution onto the section (dropping or continuously adding it).

[0033] In one embodiment, the amount of swelling agent in the swelling step is set so as to contact at least the entire upper surface of the tissue section. For example, the swelling agent is dropped onto the entire upper surface of the tissue section.

[0034] The temperature of the swelling agent in the swelling step is 35°C or higher and 45°C or lower. In one embodiment, the temperature is 37°C. By configuring it in this way, the tissue section can be swollen at a temperature close to the body temperature.

[0035] The contact time in the swelling step is preferably 16 hours or longer and 32 hours or shorter, and preferably about 24 hours. By configuring it in this way, the tissue section can be gradually swollen over a long period of time.

[0036] Preferably, the swelling step is carried out in an environment with a humidity such that the tissue section and the swelling agent do not dry out. In one embodiment, the swelling step is carried out in a wet box containing an appropriate amount of water.

[0037] As described above, in the swelling step, the tissue section is gently swollen. (Transfer step) In the transfer step, after bringing a transfer agent into contact with the tissue section, the tissue section is heated in a state where the tissue section is in contact with an analysis plate, and a part of the tissue section is transferred to the analysis plate. More specifically, in the transfer step, the tissue section is pressed against the analysis plate and heated in a state where the transfer agent is in contact with at least the upper surface of the tissue section. By the transfer step, a part of the tissue section is transferred to the analysis plate as a transfer product.

[0038] As described above, the tissue section is typically mounted on the primary plate. Therefore, in the transfer step, typically, after bringing the transfer agent into contact with the tissue section, the tissue section is heated in a state where the primary plate and the analysis plate are overlapped so that the tissue section comes into contact with the analysis plate, and a part of the tissue section is transferred to the analysis plate.

[0039] In one embodiment, in the transfer step, the analysis plate is overlapped on the primary plate so that the tissue section faces the analysis plate. Thereby, due to the self-weight of the analysis plate, the surface (lower surface) of the analysis plate is pressed against the upper surface (transfer surface) of the tissue section. Thereby, the surface of the analysis plate is adhered to the upper surface of the tissue section. As long as the transfer step can be appropriately carried out, the surface of the analysis plate may be strongly pressed against the upper surface of the tissue section by pressing the analysis plate downward.

[0040] In other embodiments, the primary plate may be placed over the analysis plate such that the tissue section faces the analysis plate.

[0041] The transfer agent contains ammonium bicarbonate, acetonitrile, and water. In one embodiment, the concentration of ammonium bicarbonate in the transfer agent is 0.05 M or more and 0.15 M or less, and the concentration of acetonitrile in the transfer agent is 20% or more and 70% or less based on the transfer agent. The remaining component in the transfer agent is water (H ２ O). If the transfer agent is configured in this way, the transfer process can be carried out efficiently.

[0042] The concentration of ammonium bicarbonate in the transfer agent is preferably 0.08 M or more and 0.12 M or less, and more preferably about 0.10 M.

[0043] The concentration of acetonitrile in the transfer agent is preferably 50% or more and 70% or less, and more preferably about 60%. By making the concentration of acetonitrile in the transfer agent higher than the concentration of acetonitrile in the swelling agent, elution of components from the tissue section is more promoted in the transfer process. As a result, in the transfer process, elution of components from the tissue section occurs vigorously in a short time.

[0044] In one embodiment, the amount of the transfer agent in the transfer process is set to at least contact the entire upper surface of the tissue section. For example, the transfer agent is dropped onto the entire upper surface of the tissue section.

[0045] The components eluted in the transfer agent adhere to the analysis plate that has contacted the tissue section. It is preferable that the heating in the transfer process is carried out until the transfer agent outside the tissue section disappears. More specifically, by completely evaporating the transfer agent near the upper surface of the tissue section, it is possible to suppress the elution of the components adhered to the analysis plate back into the transfer agent and peeling off. That the transfer agent near the upper surface of the tissue section has been substantially evaporated can be confirmed by visually confirming that the transfer agent outside the tissue section (around the tissue section) has disappeared.

[0046] The heating temperature in the transfer process is preferably 80°C to 110°C, more preferably 90°C to 100°C, and in one embodiment, the heating temperature was 94°C. By raising the temperature to a high temperature close to 100°C, the transfer agent can be brought to a boil, accelerating the evaporation rate of the transfer agent. In addition, the cross-linked structure of the protein that was cross-linked during paraffin embedding is destroyed by the high temperature, making it easier to mix the protein with the matrix material and making it easier for the protein to be ionized.

[0047] The heating method for the transfer process is not particularly limited. For example, the tissue sections may be heated to the above temperature by heating the air around the primary plate and analysis plate, with the tissue sections sandwiched between them, using an oven set to the above temperature. Alternatively, the tissue sections may be heated to the above temperature by heat conduction from the analysis plate by bringing a metal plate set to the above temperature into contact with the analysis plate. Furthermore, if it is expected that the tissue sections will be several degrees lower than the set temperature of the oven and metal plate even when using an oven and metal plate, the set temperature of the oven and metal plate may be set several degrees higher than the above temperature.

[0048] In one embodiment, the primary plate and analysis plate, with the tissue sections sandwiched between them, are placed in a hybridizer heated to 94°C and heated. At this time, the tissue sections are heated to a temperature of 94°C or close to 94°C (for example, a temperature a few degrees lower than 94°C).

[0049] In the above example, the method described involves contacting the tissue section with the transfer agent, then contacting the tissue section with the analysis plate, and finally heating the tissue section. However, the temporal order of "contact of the tissue section with the transfer agent," "contact of the tissue section with the analysis plate," and "heating of the tissue section" may be adjusted within the range that achieves the effects of this embodiment. In other words, there should be a moment when the upper surface of the tissue section is pressed against the high-temperature analysis plate while the transfer agent is present on the upper surface of the tissue section. For example, "contact of the tissue section with the transfer agent," "contact of the tissue section with the analysis plate," and "heating of the tissue section" may be started simultaneously. However, it is preferable that "contact of the tissue section with the transfer agent" is performed prior to "heating of the tissue section" in order to allow time for the transfer agent to permeate the upper surface of the tissue section and for the components to dissolve in the transfer agent.

[0050] (Analysis Plate) In this specification, an analysis plate is a sample plate used in a desired analysis of a tissue section.

[0051] In one embodiment, the analysis plate is made of transparent conductive film (ITO: Indium Tin Oxide) glass. With this configuration, it is possible to perform mass spectrometry on the transfer material attached to the analysis plate. Furthermore, because it is transparent, it is easy to confirm through the ITO glass that the transfer agent between the primary plate on which the sample was originally placed and the ITO glass has evaporated.

[0052] In addition to metal plates, glass plates, and heat-resistant resin plates, the appropriate plate is selected depending on the type of analysis and the component being analyzed.

[0053] (Effects of this embodiment) As described above, the tissue section transfer method according to this embodiment allows the section to be transferred to a general-purpose analysis plate.

[0054] This offers the following advantages. Firstly, in research institutions, hospitals, etc., preserved sections are often fixed on non-conductive primary plates (typically glass slides). However, with the exception of some specialized mass spectrometers, general mass spectrometers can only analyze samples on conductive analytical plates. Therefore, in research institutions, hospitals, etc., it is not possible to perform mass spectrometry on preserved sections using general mass spectrometers. According to this embodiment, since the sections can be transferred to a metal plate or a conductive non-metal plate, it becomes possible to analyze the components of the preserved sections using a general mass spectrometer. This means that, for example, sections collected and preserved in an era before mass spectrometers existed can also be analyzed using a general mass spectrometer.

[0055] Secondly, according to this embodiment, tissue sections can be transferred to a general-purpose analysis plate. This reduces the cost and effort required to prepare special analysis plates.

[0056] Thirdly, according to this embodiment, the swelling agent and the transfer agent are very simple and inexpensive aqueous solutions consisting of ammonium bicarbonate, acetonitrile, and water.

[0057] As shown in the first to third examples, according to this embodiment, sections can be transferred to an analysis plate by a very inexpensive and simple method.

[0058] Fourthly, since the transcript of the section can be used as the analysis target, the desired analysis can be performed while preserving the original section. This makes it possible to perform retrospective analyses, such as mass spectrometry, on samples that were observed and preserved in the past. Furthermore, the original section can be observed and analyzed in the same way as in the past. In some cases, the original section can be subjected to an analysis different from the one performed on the transcript of that section.

[0059] Furthermore, this embodiment can also be applied to untreated sections. Even when this embodiment is applied to untreated sections, it is clear that the transfer efficiency is improved compared to simply pressing the untreated sections onto an analysis plate.

[0060] Fifth, according to this embodiment, multiple transfers can be created from a single section. Specifically, for example, when the inventors pressed a single section, which had been in contact with a transfer agent, onto five analysis plates under high-temperature conditions during the transfer process, a transfer was generated on each of the five analysis plates. This means that multiple types of analysis can be performed on each of the multiple transfers derived from a single section.

[0061] For example, when performing mass spectrometry (MALDI-MS) using matrix-assisted laser desorption / ionization (MALDI), the corresponding matrix solution differs depending on the component to be analyzed. Therefore, if you want to analyze a first type of component and a second type of component, and there is only one transfer from a single section, then depositing the matrix solution corresponding to the first type of component may prevent the analysis of the second type of component. According to this embodiment, since multiple transfers are obtained from a single section, MALDI analysis of multiple types of components in a single section becomes possible.

[0062] Furthermore, for each of the multiple transcripts derived from a single section, it is possible to perform protein analysis by MALDI-MS and elemental analysis by inductively coupled plasma mass spectrometry (ICP-MS).

[0063] Furthermore, it is possible to perform both mass spectrometry and non-mass spectrometry analyses on each of the multiple transcripts derived from a single section, and it is also possible to perform multiple types of non-mass spectrometry analyses.

[0064] [Additional steps] As shown in Figures 1 and 2, the method for transferring tissue sections according to this embodiment may include additional steps.

[0065] (Deparaffinization process) As described above, a deparaffinization process is performed on paraffin-embedded sections before the swelling process.

[0066] In the paraffin removal process, first, the sections are repeatedly immersed in xylene to remove the paraffin. Next, the sections are repeatedly immersed in 100% ethanol to remove the xylene. Furthermore, the sections are immersed in ethanol of a lower concentration, and then washed with water to remove the ethanol.

[0067] Furthermore, drying the sections removes water from them, making it easier for the swelling agent to penetrate the tissue during the swelling process.

[0068] In one embodiment, during the deparaffinization process, the paraffin-embedded tissue section on a glass slide (paraffin-embedded section) is immersed in xylene for 5 minutes, and this is repeated three times. Then, the tissue section is immersed in 100% ethanol three times and in 70% ethanol approximately three times. After removing the 70% ethanol from the tissue section, the entire tissue section is washed with sterile water at room temperature to remove the ethanol component. Finally, the tissue section is allowed to air dry for 1 hour.

[0069] Tissue sections that have undergone a deparaffinization process after being paraffin-embedded are not covered with paraffin, just like fresh frozen sections. As a result, tissue sections that have undergone a deparaffinization process after being paraffin-embedded can be transferred to a general-purpose analytical plate using the transfer method according to this embodiment, just like fresh frozen sections.

[0070] (Digestion Step) The method for transferring tissue sections according to the embodiment may further include a digestion step in which a digesting agent containing a proteolytic enzyme is added to the tissue section between the swelling step and the transfer step. In the digestion step, the proteins in the tissue section are digested (fragmented) and converted into peptides by the proteolytic enzyme. Therefore, mass spectrometry can be performed on the transcript of the tissue section that has undergone the digestion step to obtain a mass spectrum of peptides, which is useful for identifying the proteins contained in the tissue section. On the other hand, the digestion step does not need to be performed when imaging is to be performed with the proteins in an intact state. Also, the digestion step does not need to be performed when the component to be analyzed is not a protein. In one example, the proteolytic enzyme and the digesting agent are trypsin and trypsin solution.

[0071] In the digestion process, a protein digestant is prepared, for example, by diluting a commercially available trypsin solution. The digestion process is then carried out with the digestant in contact with the entire upper surface of the tissue section. The contact time in the digestion process is, for example, 24 hours. The temperature of the digestant in the digestion process is, for example, 37°C.

[0072] (Matrix Addition Step) The tissue section transfer method according to this embodiment may further include a matrix addition step in which a matrix solution containing a matrix substance is added to the transfer on the analysis plate. This allows MALDI-MS to be performed on the transfer on the analysis plate. MALDI-MS is easy to perform and allows for rapid analysis. Furthermore, by performing MALDI imaging, the two-dimensional distribution of the analyte can be confirmed.

[0073] Specifically, for example, MALDI-MS pretreatment can be performed by spraying a matrix solution containing a matrix substance over the entire transfer on an analysis plate. Examples of matrix substances include α-cyano-4-hydroxycinnamic acid (4-CHCA), α-cyano-3-hydroxycinnamic acid (3-CHCA), sinapic acid, ferulic acid, 3-hydroxy-4-nitrobenzoic acid (3H4NBA), 2,5-dihydroxybenzoic acid, 1,5-diaminonaphthalene, or 9-aminoacridine (9-AA).

[0074] (Mass Spectrometry) An analysis plate containing a matrix material is placed in a mass spectrometer. Laser light (e.g., Nd:YAG laser) is shone onto the transcription material, ionizing the peptides in the transcription material into peptide ions, which are then detected by a detector. The detection results are transmitted to a processing unit, which generates a mass spectrum for each transcription material. The processing unit also performs mass spectrometry imaging, representing the intensity of the ion detection signal corresponding to a predetermined m / z value in relation to its position on the analysis plate.

[0075] Here, the processing device is, for example, a computer, and includes a processor such as a CPU, a memory, a display, and an input device such as a mouse.

[0076] The user can confirm the components in the section by checking the mass spectrum and mass spectrometry imaging results displayed on the display.

[0077] (Components to be analyzed) According to the tissue section transfer method according to the above embodiment, fresh frozen sections are suitable for the transfer of components of proteins, peptides, low molecular weight metabolites, and lipids. In addition, paraffin-embedded sections in which at least a part of low molecular weight metabolites and lipids flow out in the deparaffinization step are suitable for the transfer of components of proteins and peptides.

[0078] As described above, by analyzing the proteins, peptides, low molecular weight metabolites, and / or lipids contained in the transcript of the tissue section according to the present embodiment, metabolomics that comprehensively analyzes these metabolites is also possible. In addition, by analyzing the transcript of pathological tissue, it can also be used for disease research and diagnosis.

[0079] [Example] In this example, a fresh frozen section of a mouse kidney mounted on a slide glass was used as the tissue section to which the transfer method according to the present embodiment was applied. For each reagent in this example, unless otherwise specified, the remaining components other than the described components were H ２ O.

[0080] (1) Swelling step 100 μL of a swelling agent (0.1 M NH ４ HCO ３ , 30% CH ３ CN) was dropped onto the entire tissue section on the slide glass, and the tissue section was transferred to a wet box containing an appropriate amount of water and incubated at 37 °C for 24 hours.

[0081] (2) The digestion tissue sections were allowed to return from 37°C to room temperature and the swelling agent was removed. Trypsin Platinum, Mass Spectrometry Grade (Promega, Madison, Wisconsin) was dissolved in 50 mM acetic acid to prepare a trypsin solution (0.5 μg / μL). 10 μL of this trypsin solution was diluted with a diluent (25 mM NH₄). ４ HCO ３ , 10% CH ３ A digestant was diluted 10-fold with 90 μL of CN to a trypsin concentration of 0.05 μg / μL, and 100 μL of this digestant was dropped onto the entire tissue section on a glass slide. Then, the glass slides containing the tissue sections were transferred to a humid chamber filled with water to a depth of approximately 1 cm and incubated at 37°C for 24 hours.

[0082] (3) Transfer process: The slide glass containing the tissue section was returned from 37°C to room temperature (approximately 23°C), and the trypsin solution was removed from the slide specimen. Then, the transfer solution (0.1 M NH₄) was used. ４ HCO ３ , 60% CH ３ 50 μL of CN was dropped onto the entire tissue section on a glass slide. Next, the conductive side of an ITO glass was pressed onto the tissue section from above, and the area was heated using a hybridizer (Programmable Hybridizer S2450, manufactured by Dako, Glostrup, Denmark) set to 94°C until the dropped transfer solution had completely evaporated.

[0083] After heating, the microscope slide and ITO glass containing the tissue sections were rapidly cooled on an aluminum rack at 4°C for 5 minutes. The microscope slide and ITO glass were then separated. This created a transfer on the ITO glass. Hereafter, the tissue sections remaining on the microscope slide will also be referred to as the "original tissue sections."

[0084] (4) Matrix addition process A matrix solution (containing 9-AA) was deposited to a thickness of 0.9 μm over the entire transfer on the ITO glass using a matrix deposition apparatus (iMLayer manufactured by Shimadzu Corporation). Similarly, a matrix solution of the same composition was deposited over the entire original tissue section on the glass slide.

[0085] (5) Mass spectrometry mass spectrometer (“iMScope”) ＴＭ Using QT, the transfer material on ITO glass was analyzed at laser irradiation diameter settings of 2 (approximately 25 μm) and 4 (approximately 50 μm). For comparison, the original tissue section was also transferred to another ITO glass and analyzed similarly. After confirming whether the peak of bio-derived phospholipids was detected in both the original tissue section and the transfer material, mass spectra were obtained, and mass spectrometry imaging corresponding to multiple m / z values ​​was acquired.

[0086] Figure 3 shows the mass spectrum of the transcript. Figure 4 shows the mass spectrum of the original tissue section. Referring to Figures 3 and 4, the mass spectrum of the transcript showed the same pattern as the mass spectrum of the original tissue section. As described above, the same components as those found in the original tissue section were detected in the transcript.

[0087] Figure 5 shows the results of mass spectrometry imaging of the transcript and the original tissue section. The upper panel of Figure 5 shows the optical image and mass spectrometry imaging results of the original tissue section. The lower panel of Figure 5 shows the optical image and mass spectrometry imaging results of the transcript. More specifically, the lower panel of Figure 5 shows the optical image and mass spectrometry imaging results of a portion of the transcript corresponding to a part of the original tissue section (the area enclosed by a rectangle).

[0088] The leftmost column of Figure 5 shows optical images acquired with an optical microscope using a 5x objective lens. Referring to the optical images, it was observed that the original tissue section was entirely transferred to the ITO glass target. The three rightmost columns of Figure 5 show the results of mass spectrometry imaging for three different substances corresponding to m / z 885.54, m / z 904.61, and m / z 906.61, respectively. Referring to the mass spectrometry imaging results, the distribution of each of the three substances was similar in both the transfer and the original tissue section. This indicates that the distribution of the specified substances in the original tissue section was reflected in the transfer.

[0089] [Aspects] The above-described exemplary embodiments will be understood by those skilled in the art to be specific examples of the following aspects.

[0090] (Section 1) A method for transferring tissue sections according to one embodiment includes a swelling step of bringing a swelling agent into contact with a tissue section mounted on a primary plate, and a transfer step of bringing a transfer agent into contact with the tissue section, and then heating the tissue section with the primary plate and the analysis plate superimposed so that the tissue section is in contact with the analysis plate, thereby transferring a portion of the tissue section to the analysis plate. The swelling agent and the transfer agent each contain ammonium bicarbonate, acetonitrile, and water.

[0091] According to the tissue section transfer method described in paragraph 1, sections such as paraffin-embedded sections and fresh frozen sections can be transferred to a general-purpose analytical plate.

[0092] (Section 2) In the method for transferring tissue sections described in Section 1, the concentration of ammonium bicarbonate in the swelling agent is 0.05 M or more and 0.15 M or less. The concentration of acetonitrile in the swelling agent is 20% or more and 70% or less based on the swelling agent.

[0093] According to the tissue section transfer method described in paragraph 2, the transfer process can be carried out efficiently. (Paragraph 3) In the tissue section transfer method described in paragraph 1 or 2, the concentration of ammonium bicarbonate in the transfer agent is 0.05 M or more and 0.15 M or less. The concentration of acetonitrile in the transfer agent is 20% or more and 70% or less based on the transfer agent.

[0094] According to the tissue section transfer method described in paragraph 3, the transfer process can be carried out efficiently. (Paragraph 4) In the tissue section transfer method described in any one of paragraphs 1 to 3, the heating temperature in the transfer process is 80°C or higher and 110°C or lower.

[0095] According to the tissue section transfer method described in Section 4, the transfer agent can be brought to a boil to accelerate its evaporation rate. Furthermore, the cross-linked structure of proteins that were cross-linked during paraffin embedding is destroyed by high temperature, making it easier for the proteins to mix with the matrix material and for the proteins to be ionized.

[0096] (Article 5) In the method for transferring tissue sections described in any one of paragraphs 1 to 4, the heating in the transfer step shall be carried out until the transfer agent outside the tissue section is gone.

[0097] According to the tissue section transfer method described in Section 5, by completely evaporating and eliminating the transfer agent near the upper surface of the tissue section, it is possible to suppress the leaching of components attached to the analysis plate back into the transfer agent and causing them to detach.

[0098] (Item 6) In the method for transferring tissue sections described in any one of items 1 to 5, the temperature of the swelling agent in the swelling step is 35°C or higher and 45°C or lower.

[0099] According to the method for transferring tissue sections described in paragraph 6, the tissue sections can be swollen at a temperature close to that inside the body.

[0100] (Section 7) In the method for transferring tissue sections described in any one of Sections 1 to 6, the contact time for the swelling step is 16 hours or more and 32 hours or less.

[0101] According to the method for transferring tissue sections described in Section 7, the tissue sections can be gradually swollen over a long period of time.

[0102] (Clause 8) In the method for transferring tissue sections described in any one of paragraphs 1 to 7, the tissue section is a tissue section that has been deparaffinized from a paraffin-embedded section.

[0103] According to the tissue section transfer method described in Section 8, a portion of the paraffin-embedded section, which is the most typical preserved section, can be transferred to an analysis plate and subjected to analysis.

[0104] (Paragraph 9) In the method for transferring tissue sections described in any one of paragraphs 1 to 7, the tissue sections are fresh frozen sections.

[0105] According to the tissue section transfer method described in paragraph 9, a portion of fresh frozen sections, which are typical preserved sections that, together with paraffin-embedded sections, constitute the majority of preserved sections, can be transferred to an analysis plate and subjected to analysis.

[0106] (Section 10) The method for transferring tissue sections described in any one of paragraphs 1 to 9 further includes a digestion step in which a digestive agent containing a proteolytic enzyme is added to the tissue section between the swelling step and the transfer step.

[0107] In the digestion step of the tissue section transcription method described in paragraph 10, the proteins in the tissue section are digested (fragmented) and converted into peptides by proteolytic enzymes. Therefore, mass spectrometry of the transcript of the tissue section that has undergone the digestion step will yield a peptide mass spectrum, which is useful for identifying the proteins contained in the tissue section.

[0108] (Clause 11) The method for transferring tissue sections described in any one of paragraphs 1 to 10 further comprises a matrix addition step of adding a matrix solution containing a matrix substance to a portion of the tissue sections transferred to an analysis plate.

[0109] According to the tissue section transfer method described in paragraph 11, MALDI-MS can be performed on the transfer material on the analysis plate.

[0110] The embodiments disclosed herein should be considered in all respects to be illustrative and not restrictive. The scope of the invention is indicated by the claims rather than the foregoing description, and all modifications within the meaning and scope of equivalents of the claims are intended.

Claims

1. A method for transferring tissue sections, comprising: a swelling step of bringing a swelling agent into contact with tissue sections mounted on a primary plate; and a transfer step of bringing a transfer agent into contact with the tissue sections, and then heating the tissue sections while the primary plate and the analysis plate are stacked so that the tissue sections are in contact with the analysis plate, thereby transferring a portion of the tissue sections to the analysis plate, wherein the swelling agent and the transfer agent each comprise ammonium bicarbonate, acetonitrile, and water.

2. The method for transferring tissue sections according to claim 1, wherein the concentration of ammonium bicarbonate in the swelling agent is 0.05 M or more and 0.15 M or less, and the concentration of acetonitrile in the swelling agent is 20% or more and 70% or less based on the swelling agent.

3. The method for transferring tissue sections according to claim 1 or 2, wherein the concentration of ammonium bicarbonate in the transfer agent is 0.05 M or more and 0.15 M or less, and the concentration of acetonitrile in the transfer agent is 20% or more and 70% or less based on the transfer agent.

4. The method for transferring tissue sections according to claim 1 or 2, wherein the heating temperature in the transfer step is 80°C or higher and 110°C or lower.

5. The method for transferring tissue sections according to claim 1 or 2, wherein the heating in the transfer step is carried out until the transfer agent outside the tissue section is gone.

6. The method for transferring tissue sections according to claim 1 or 2, wherein the temperature of the swelling agent in the swelling step is 35°C or higher and 45°C or lower.

7. The method for transferring tissue sections according to claim 1 or 2, wherein the contact time for the swelling step is 16 hours or more and 32 hours or less.

8. The method for transferring tissue sections according to claim 1 or 2, wherein the tissue section is a tissue section obtained by deparaffinizing a paraffin-embedded section.

9. The method for transferring tissue sections according to claim 1 or 2, wherein the tissue sections are fresh frozen sections.

10. A method for transferring tissue sections according to claim 1 or 2, further comprising a digestion step of adding a digestive agent containing a proteolytic enzyme to the tissue section between the swelling step and the transfer step.

11. The method for transferring tissue sections according to claim 1 or 2, further comprising a matrix addition step of adding a matrix solution containing a matrix material to a portion of the tissue section transferred to the analysis plate.

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