EP4630780A1 - Method for an automated tissue preserving histopathologic analysis of biospecimens - Google Patents

Method for an automated tissue preserving histopathologic analysis of biospecimens

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Publication number
EP4630780A1
EP4630780A1 EP24837564.4A EP24837564A EP4630780A1 EP 4630780 A1 EP4630780 A1 EP 4630780A1 EP 24837564 A EP24837564 A EP 24837564A EP 4630780 A1 EP4630780 A1 EP 4630780A1
Authority
EP
European Patent Office
Prior art keywords
tissue
biospecimens
container
tissue sample
preserving
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24837564.4A
Other languages
German (de)
French (fr)
Inventor
René HÄGERLING
Fabian Mohr
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Limaa Technologies GmbH
Original Assignee
Limaa Technologies GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Limaa Technologies GmbH filed Critical Limaa Technologies GmbH
Publication of EP4630780A1 publication Critical patent/EP4630780A1/en
Pending legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36—Embedding or analogous mounting of samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • G—PHYSICS
    • G06—COMPUTING OR CALCULATING; COUNTING
    • G06V—IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING
    • G06V20/00—Scenes; Scene-specific elements
    • G06V20/60—Type of objects
    • G06V20/64—Three-dimensional [3D] objects
    • G—PHYSICS
    • G06—COMPUTING OR CALCULATING; COUNTING
    • G06V—IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING
    • G06V20/00—Scenes; Scene-specific elements
    • G06V20/60—Type of objects
    • G06V20/69—Microscopic objects, e.g. biological cells or cellular parts
    • G06V20/693—Acquisition
    • G—PHYSICS
    • G06—COMPUTING OR CALCULATING; COUNTING
    • G06V—IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING
    • G06V20/00—Scenes; Scene-specific elements
    • G06V20/60—Type of objects
    • G06V20/69—Microscopic objects, e.g. biological cells or cellular parts
    • G06V20/698—Matching; Classification

Definitions

  • Histopathological analysis of tissue specimens is a multi-step process performed in research and development or clinical pathology laboratories. These steps include, among other things, embedding of the sample in paraffin, dehydration, physical sectioning of tissue blocks, transfer to glass slides, staining, sealing, microscopy and/or digitization of sample slides using slide scanners.
  • steps include, among other things, embedding of the sample in paraffin, dehydration, physical sectioning of tissue blocks, transfer to glass slides, staining, sealing, microscopy and/or digitization of sample slides using slide scanners.
  • a full automation of the entire process is currently not or only to a limited degree possible as the glass slide based histology approach has intrinsic limitation in regards of preparation time, quality, completeness of the tissue sample and abilities to perform multiplex marker stainings.
  • Optical tissue clearing presents a suitable method for multiplex staining of entire tissue specimens.
  • the specimen becomes translucent, and therefore allows light to penetrate the tissue and consequently increase the accessible and imaging depth for optical sectioning.
  • Available methods for optical clearing and multiplex tissue staining require expensive reagents and time-consuming incubation steps.
  • the speed and degree of optical transparency varies depending on tissue or organ type as well as pathological entity. To increase the interpretability of such images by trained medical specialist in the field of histopathology an automated approach for optical tissue clearing with high transparency and speed is required.
  • US 2020/0209118 A1 disclosed the aforementioned approach for tissue preparation and imaging of entire tissue specimens. It discloses a method for preparing an animal tissue for fluorescence microscopy that allows visualization of single cells within mammalian tissues. This document discloses the technological background of visualization of single cells within tissue samples but does not disclose a histopathology analysis of biospecimens suitable for high number of tissue samples currently processed in clinical pathology laboratories.
  • US 10,591 ,392 B2 discloses a method of processing biopsy sized tissue samples using fluorescence microscopy. However, the disclosed method is not automated and bases on manual processing of the tissue samples.
  • the method for an automated tissue preserving histopathologic analysis of biospecimens comprises at least the steps of: a) obtaining a tissue sample; b) storing the tissue sample in a tissue container, wherein the tissue container includes an identification tag; c) filling the tissue container with a dehydrating fluid for a first time-period and draining the dehydrating fluid after the first time-period; d) filling the tissue container with at least one labelling fluid for a second timeperiod and draining the at least one labelling fluid after the second time-period; e) filling the tissue container with a clearing solution; f) imaging the tissue sample in the tissue container using a 3D imaging device, particularly a light-sheet microscope; g) processing the image obtained using the 3D imaging device by: h) identifying and quantifying labelled structures, like a cell, a cellular component, cells, cell types and/or extracellular content and tissue architecture in its spatial distribution; and i) visualizing the analysed tissue sample
  • the inventive method starts with receiving a tissue sample.
  • the tissue sample is a biospecimen, which has been extracted from a human or animal, for example during a minimal-invasive surgery, an endoscopic procedure, a fine needle biopsy or similar procedure for extracting a biospecimen from a human or animal.
  • biospecimen and tissue sample can be used interchangeably.
  • the tissue sample can consist of multiple separate biospecimen which have been obtained individually from the same human or animal and which are processed together as a single tissue sample.
  • the tissue sample is a fixed tissue sample.
  • the step of fixing the biospecimen extracted from the human or animal body is not necessarily covered by the inventive method.
  • the extracted biospecimen is for example fixed by treating the biospecimen with a fixing solution, particularly by immersing the biospecimen in the fixing solution.
  • the biospecimen is kept in the fixing solution until it has been fixed.
  • the fixing solution is for example Formalin or 4 % PFA.
  • Fixing the tissue sample refers to any process that halts cellular degradation and respectively a fixed tissue sample is not subject to cellular tissue degradation.
  • the fixed tissue sample can be washed prior to further processing with a liquid for removing the fixing solution.
  • a tissue sample fixed using formalin can be washed using a phosphate buffered saline.
  • tissue sample that is not fixed, like for example a direct (on the fly) analysis after obtaining the tissue sample from the human or animal body.
  • the obtained tissue sample is stored in a tissue container for further processing.
  • the tissue container comprises an identification tag, so that the tissue container and hence the contained tissue sample, can be tracked throughout the whole process of the invention.
  • a tracking of the tissue samples is an essential feature of the automated histopathologic analysis because the method is performed without human interaction and there must be some means for checking the status of the processing of the single tissue samples. This requires a tracking of the tissue container throughout the whole processing.
  • the tissue container is filled with a dehydrating fluid for a first time-period and the dehydrating fluid is drained after the first time-period (referred to as step c) of the method according to the invention).
  • the dehydrating fluid is used for dehydrating the tissue sample in the tissue container and making it miscible with the clearing fluid used during step e).
  • Dehydrating refers to the removal of water and/or (partially) lipids from the tissue sample, which aids in preparation of the tissue sample for the later imaging of the tissue sample during step f) of the method.
  • the dehydrating has the advantage that in the later process an anhydrous clearing reagent or a clearing fluid with low water solubility can be used during step e).
  • the first time-period is chosen such that the tissue sample is sufficiently dehydrated and hence depends on the size of the respective tissue sample and type of the tissue sample. Considering common tissue sample sizes between 0,1 cm and 3 cm the first time-period will be between 15 seconds and 1 hour. However, this might differ for different types of tissue samples like lung or lymph nodes.
  • the dehydrating fluid is preferably a water-miscible anhydrous fluid.
  • the dehydrating fluid is chosen e.g. from methacarn or alcohols such as methanol, ethanol and propanol.
  • Step c) of the method can also be repeated two or more times using the same or another dehydrating fluid, particularly in different concentrations.
  • the tissue container is filled with at least one labelling fluid for a second time-period and the at least one labelling fluid is drained after the second time-period.
  • the terms colouring liquid, labelling liquid and marking liquid can be used synonymously.
  • the labelling fluid is used for labelling one or more target molecules of the tissue sample contained in the tissue container.
  • the labelling fluid is for example a fluorescent dye.
  • the labelling fluid bases on antibodies, preferably nanobodies (VHHs, single domain antibodies), as labelling agents, wherein nanobodies are significantly smaller than antibodies and hence penetrate the tissue sample more easily.
  • the antibodies and/or nanobodies are preferably labelled using chemical or biological compounds, like for example fluorescent dyes. If antibodies respectively nanobodies are used as labelling fluid in step d), this step d) can also be performed before step c). If the labelling fluid is a fluorescent chemical, step d) is performed after step c). A combination of both can also been performed. In that case performing step d) before step c) is preferred.
  • the second-time period is chosen such that the labelling fluid can penetrate the whole tissue sample contained in the tissue container and labelling agents of the labelling fluid can bind to the one or more target molecules.
  • the tissue container is filled with a clearing solution.
  • the clearing solution is used for optically clearing the tissue sample in the tissue container. Optically clearing refers to matching the refractive index within the tissue sample, enabling (fluorescence) imaging deep within the tissue sample during the following step f) of the method according to the invention.
  • the clearing solution is for example chosen from water-based or non-water-based solvents, e.g. inorganic solvents. In case the clearing solution is a water-based chemical, the dehydration step c) is optional and can be omitted.
  • Step e) of the method can also be repeated two or more times using the same or another clearing solution, particularly in different concentrations.
  • Step f) of the method according to the invention refers to imaging the tissue sample in the tissue container using a 3D imaging device, particularly a light-sheet microscope.
  • a 3D imaging device particularly a light-sheet microscope.
  • suitable 3D imaging devices can be used, like e.g. a handheld MRT device, Multiphoton microscope, confocal or fluorescence microscope or optical projection tomograph.
  • the 3D imaging provides a 3D visualization of the (internal) macro- and microstructures in the tissue sample contained in the tissue container, preferably at sub-micron to nanoscale spatial resolution.
  • a light-sheet microscope uses a thin sheet of light to excite only fluorophores within the focal volume and scans the whole tissue sample sheet by sheet (optical section by optical section).
  • a lightsheet microscope has a true optical sectioning capability and, hence, provides sufficient axial resolution in all directions, e.g. isotropic point-spread function, restrict photobleaching and phototoxicity to a fraction of the sample and use cameras to record tens to thousands of images per second.
  • the image obtained using the 3D imaging device is processed in step g) of the method of the present invention.
  • the labelled structures are identified and quantified.
  • the labelled structures refer to a cell, a cellular component, cells, cell types and/or extracellular content and tissue architecture in its spatial distribution.
  • a cellular component refers inter alia to a cell nucleus, cell nuclei, organelles, or similar.
  • the analysed tissue sample is visualized in a 3D model and/or in multiple 2D sectional views.
  • the method results in a visualization of the tissue sample obtained during step a) in a 3D model and/or in multiple 2D sectional views.
  • the labelled structures are shown in the visualization and a pathologist can examine the tissue sample based on the visualization.
  • All steps of the afore-mentioned method are executed automatically and at least steps c) to i) do not involve any human action other than optionally supervising the process.
  • the sequence of the method steps can be changed, as far as this is technically possible. Thus, as long as steps do not sequentially depend on each other, the sequence of these method steps can be changed.
  • the automation of the whole process is facilitated by using a single tissue container for one specific tissue sample throughout the whole process.
  • the processing can be supervised and controlled because of the identification tag associated with the tissue container.
  • the identification tag is preferably an electronic tag like a RFID-tag.
  • optical identification tags using for example barcodes or QR-codes.
  • the tissue container has a defined design and all tissue container handling devices, like one or more devices for filling the tissue container with the different fluids and the light-sheet microscope are adapted for handling this specific design of the tissue container. It is also possible to use tissue containers of different sizes, as long as all tissue containers have at least some areas in common, which are used for handling and/or processing the tissue contained respectively the tissue sample contained in the tissue container.
  • the tissue container and the clearing fluid used in step e) of the method have matching refractive indices. This is particularly advantageous during imaging step f) of the inventive method.
  • the method further comprises the step of: j) filling the tissue container with a permeabilization fluid for a third time-period and draining the permeabilization fluid after the third time-period, wherein this step is preferably performed between steps b) and c) and afterwards filing the tissue container with a blocking agent for a fourth time-period and draining the blocking agent after the fourth time-period.
  • the permeabilization fluid is accelerating the access of the at least one labelling fluid in step d) into the tissue sample, particularly into deeper portions of the tissue sample.
  • This step j) is particularly useful if the at least one labelling fluid comprises antibodies because the permeabilization fluid facilitates the penetration of the antibodies into the tissue sample. If the labelling fluid comprises the smaller nanobodies the step j) is not necessarily required and can be omitted for reducing the overall process time. It is to be understood that if a permeabilization solution is used in a particular step of the methods of the invention, this does not exclude that other fluids, e.g. fluids used in other steps of the method, may also contribute to the permeabilization and improve permeabilization. For instance, a fluid for the removal of heme can be a solution that contributes to the permeabilization.
  • the blocking agent is for example an aqueous bovine serum albumin solution or serum of various species.
  • the blocking agent improves the staining of sample because the blocking agent for example reduces unspecific binding of the chemical and/or labelling reagents. This increases the quality of the staining and analysis of the tissue sample.
  • the method comprises the step of: k) checking the clearing or transparency of the tissue sample in the tissue container after the second time-period.
  • This step ensures that the tissue sample in the tissue container is suitable for the imaging in step f) of the method.
  • the checking bases for example on relative transparency compared to original tissue sample.
  • the method further comprises the step of: l) recolouring the image to match the colouring of conventional histology slides, wherein this step is preferably performed between steps h) and i).
  • the 3D model and/or multiple 2D sectional views created by the inventive methods have the same appearance than probes resulting from histopathology processes based on cutting and staining tissue samples and the pathologist can analyse the 3D model and/or multiple 2D sectional views as known to him, without adapting to new appearance like colouring of the tissue samples, particularly the labelled structures, like a cell, a cellular component, cells, cell types and/or extracellular content.
  • the method according to the invention comprises the step of: m) filling the tissue container with multiple fluorescent dyes, wherein the multiple fluorescent dyes are filled simultaneously into the tissue container or by repeating step d) multiple times.
  • the multiple fluorescent dyes are used for labelling/staining different structures, like a cell, a cellular component, cells, cell types and/or extracellular content and tissue architecture in its spatial distribution. This can happen simultaneously, particularly if the labelling of the different structures using the multiple fluorescent dyes requires similar timing or and at least partially sequentially by repeating step d), if the labelling of the different structures using the multiple fluorescent dyes requires different timing.
  • the second time-period can differ for each fluorescent dye.
  • a different channel in the 3D model or in the multiple 2D sectional views is used for each fluorescent dye (staining) a different channel in the 3D model or in the multiple 2D sectional views.
  • the pathologist can visualize the different labelled/stained structures by enabling/disabling respective channels in the 3D model or in the multiple 2D sectional views. In this way the pathologist can visualize or suppress different labelled/stained structures during his analysis depending on the current needs.
  • the method comprises the step of: n) decolouring the tissue sample.
  • This step for example comprises the steps of filling the tissue container with a decolouring fluid for a fifth time-period and draining the decolouring fluid after the fifth time-period, wherein this step is preferably performed before step d).
  • the decolouring fluid is preferably an alcohol, hemolysis fluid, formamide, peroxide, acid and/or base, reducing and/or oxidizing agent or similar fluid.
  • the tissue sample can be decoloured by a physical or biological treatment like laser treatment or enzyme treatment. The decolouring of the tissue sample enhances the penetration of light waves into the tissue sample during the imaging step f), which result in better image quality. The decolouring further enables a restaining of the tissue sample.
  • the inventive method comprises the step of: o) tracking tissue container through the complete process, particularly based on the identification tag of the tissue container.
  • tissue container In an automated process it is useful to continuously track the progress of the process, i.e. where are different tissue containers, what is the current process step for each tissue container, how long until the results are available for each tissue sample, and so on.
  • the basis for this information is the continuously tracking of the tissue container through the whole process.
  • the tracking preferably bases on the identification tag of the tissue container.
  • the method comprises the additional step of: p) rehydrating the tissue sample in the tissue container.
  • the tissue sample After rehydrating the tissue sample in the tissue container, the tissue sample can be processed by downstream applications such as regular histology, protein applications, archiving and/or DNA/RNA applications.
  • the method according to the invention is tissue preserving and hence the tissue sample, although already analysed by the inventive method, can be analysed by regular histology and/or DNA/RNA applications. However, in this case the tissue sample is preferably rehydrated.
  • the method comprises the step of: q) removing the one or more staining from the tissue sample, which have been caused by the one or more fluorescent dyes.
  • tissue is transferred back to its original state as it would not have been stained before and hence can be further processed.
  • the one or more staining is removed from the tissue sample by biological, physical and/or chemical treatment, particularly using a destaining fluid or solution.
  • the destaining fluid or solution is preferably an alcohol, formamide, peroxide, acid and/or base, reducing and/or oxidizing agent or similar fluid.
  • the tissue sample can be destained by a physical or biological treatment like laser treatment.
  • the method comprises the further step of: r) controlling the automated tissue preserving histopathologic analysis of biospecimens using a central process control, wherein the central process control comprises one or more of the following information: defining kind of tissue sample, origin of tissue sample, relevant analysis to be performed, required stainings, defining time-periods for different steps of method, tracking and/or recording of process steps and the corresponding results, or similar variables and/or features of the histopathologic analysis.
  • the central process control is for example a computer system having communication interfaces to devices executing the different step of the inventive method, like a device for filling and draining one or more fluids to tissue containers, the imaging device like a light-sheet microscope, devices for handling, particularly moving, the tissue containers or similar devices.
  • the central process control preferably uses a graphical user interface for user interaction.
  • the central process control can display or forward the result of the imaging process, particularly for a pathologist for analysing the imaging result.
  • the method comprises the step of: s) agitating the tissue container during step c), d), e), j), m) and/or n).
  • tissue container improves the penetration of the respective fluid filled into the tissue container during the corresponding step with the tissue sample contained in the tissue container. This reduces the respective time-period required for treating the tissue sample with the respective fluid.
  • the agitation of the tissue container reduces the overall process time, which has a great impact during automatic mass-analysis of thousands of tissue samples, which are currently analysed manually in glass slide based histology approach.
  • the method is performed at room temperature.
  • Room temperature refers to a common temperature in a laboratory, e.g. between 15°C and 25°C.
  • the method comprises the further step of: t) checking characteristics of the fluid used during step c), d), e), j), m) and/or n) and comparing the result to the expected characteristics of the fluid used in the respective step.
  • This step is an in-process control of the liquid filled into the tissue container during the respective steps. Any of the liquid has a defined characteristic and hence, it can be checked whether the liquid container has been filled with the correct liquid. This is particularly useful, if a single device is used to fill and drain different liquids to the tissue container.
  • the method comprises the step of: u) checking the fill level of the tissue container during step c), d), e), j), m) and/or n).
  • tissue container has been filled as defined, i.e. that the tissue sample inside the tissue container is completely surrounded by the liquid filled into the tissue container. This guarantees that the tissue sample is effectively and completely treated by the liquid filled into the tissue container.
  • the fill level of one or more containers containing the one or more fluids used during steps c), d), e), j), m) and/or n) may be checked respectively monitored. Thereby it is guaranteed that sufficient amounts of these fluids are available during performing the inventive method.
  • the tissue container has a tissue sample chamber having a volume of 1000 mm 3 to 3000 mm 3 , preferably about 2000 mm 3 .
  • the tissue sample chamber is cylindrically, having a base area of 79 mm 2 and a height of 25 mm.
  • Fig. 1 a flow diagram of a method for an automated tissue preserving histopathologic analysis of biospecimens according to the present invention
  • Fig. 2 a tissue sample before step c) and after step e) of the method according to the present invention.
  • Fig. 3 the visualization process of a tissue sample according to the present invention.
  • Fig. 1 shows a flow diagram of a method for an automated tissue preserving histopathologic analysis of biospecimens according to the present invention.
  • the inventive method starts with receiving a tissue sample, also referred to as step a) of the method according to the invention.
  • This tissue sample is a biospecimen, which has been extracted from a human or animal, for example during a minimal-invasive surgery, an endoscopic procedure, a needle biopsy or similar procedure for extracting a biospecimen from a human or animal.
  • the tissue sample is a fixed tissue sample.
  • the step of fixing the biospecimen extracted from the human or animal body is not necessarily covered by the inventive method.
  • the extracted biospecimen is for example fixed by treating the biospecimen with a fixing solution, particularly by immersing the biospecimen in the fixing solution.
  • the biospecimen is kept in the fixing solution until it has been fixed.
  • the fixing solution is for example Formalin or 4 % PFA.
  • the obtained tissue sample is stored in a tissue container for further processing.
  • the tissue container comprises an identification tag, so that the tissue container and hence the contained tissue sample, can be tracked throughout the whole process of the invention.
  • the tracking of the tissue samples is an essential feature of the automated histopathologic analysis because the method is performed without human interaction and there must be some means for checking the status of the processing of the single tissue samples.
  • the tissue sample in the tissue container is prepared for 3D imaging.
  • These sequence of steps comprises the single steps of dehydrating (step c)), labelling (step d)) and clearing (step e)) the tissue sample in the tissue container.
  • the tissue container is filled with a dehydrating fluid for a first time-period and the dehydrating fluid is drained after the first time-period.
  • the dehydrating fluid is used for dehydrating the tissue sample in the tissue container and making it miscible with the clearing fluid used during step e).
  • Dehydrating refers to the removal of water and/or (partially) lipids from the tissue sample, which aids in preparation of the tissue sample for the later imaging of the tissue sample during step f) of the method.
  • the dehydrating has the advantage that in the later process an optical clearing fluid with low water solubility can be used during step e).
  • the dehydrating fluid is a water- miscible anhydrous fluid.
  • the dehydrating fluid is chosen e.g. from methacarn or alcohols such as methanol, ethanol and propanol.
  • the tissue container is filled with at least one labelling fluid for a second time-period and the at least one labelling fluid is drained after the second time-period.
  • the labelling fluid is used for labelling one or more target molecules of the tissue sample contained in the tissue container.
  • the labelling fluid is for example a fluorescent dye.
  • the labelling fluid bases on antibodies, preferably nanobodies, as labelling agents.
  • the antibodies and/or nanobodies are preferably labelled using chemical or biological compounds, like for example fluorescent dyes. If antibodies respectively nanobodies are used as labelling fluid in step d), this step d) can also be performed before step c). If the labelling fluid is a fluorescent chemical, step d) is performed after step c). In case of combining both antibodies I nanobodies and fluorescent chemical, step d) is performed before step c).
  • the tissue container is filled with a clearing solution, like for example chosen from water or water-based inorganic solvents.
  • the clearing solution is used for optically clearing the tissue sample in the tissue container.
  • Optically clearing refers to matching the refractive index within the tissue sample, enabling fluorescence imaging deep within the tissue sample during the following step f) of the method according to the invention.
  • the next step f) of the method according to the invention refers to imaging the tissue sample in the tissue container using a 3D imaging device, particularly a light-sheet microscope.
  • a 3D imaging device particularly a light-sheet microscope.
  • suitable 3D imaging devices can be used, like e.g. a handheld MRT device, Multiphoton microscope, confocal or fluorescence microscope or optical projection tomograph.
  • the 3D imaging provides a 3D visualization of the internal microstructures in the tissue sample contained in the tissue container, preferably at sub-micron to nanoscale spatial resolution.
  • step g) of the method according to the invention the images obtained during step f) are processed.
  • step h) of the processing the labelled structures are identified and quantified.
  • the labelled structures refer to a cell, a cellular component, cells, cell types and/or extracellular content and tissue architecture in its spatial distribution.
  • a cellular component refers inter alia to a cell nucleus, cell nuclei, organelles, or similar.
  • step i) of the processing the analysed tissue sample is visualized in a 3D model and/or in multiple 2D sectional views.
  • the method results in a visualization of the tissue sample obtained during step a) in a 3D model and/or in multiple 2D sectional views.
  • the labelled structures are shown in the visualization and a pathologist can examine the tissue sample based on the visualization.
  • Fig. 2 shows a tissue sample before step c) and after step e) of the method according to the present invention.
  • Fig. 2 shows the obtained tissue sample before and after preparing the tissue sample during steps c), d) and e).
  • Fig. 2 visualizes the preparation of the tissue sample for the imaging.
  • Fig. 3 shows the visualization process of a tissue sample according to the present invention.
  • the images obtained e.g. by a light-sheet microscope are for example processed sheet by sheet, i.e. the input is 2D image (sheet).
  • the sheets obtained during imaging are processed individually and at the end are visualized together in a 3D visualization of an image stack.

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Abstract

The invention relates to a method for an automated tissue preserving histopathologic analysis of biospecimens comprising the steps of: a) obtaining a tissue sample; b) storing the tissue sample in a tissue container, wherein the tissue container includes an identification tag; c) filling the tissue container with a dehydrating fluid for a first time-period and draining the dehydrating fluid after the first time-period; d) filling the tissue container with at least one labelling fluid for a second time-period and draining the at least one labelling fluid after the second time-period; e) filling the tissue container with a clearing solution; f) imaging the tissue sample in the tissue container using a 3D imaging device, particularly a light-sheet microscope; and g) processing the image obtained using the 3D imaging device by: h) identifying and quantifying labelled structures, like a cell, a cellular component, cells, cell types and/or extracellular content and tissue architecture in its spatial distribution; and i) visualizing the analysed tissue sample in a 3D model and/or in multiple 2D sectional views.

Description

Method for an automated tissue preserving histopathologic analysis of biospecimens
Histopathological analysis of tissue specimens is a multi-step process performed in research and development or clinical pathology laboratories. These steps include, among other things, embedding of the sample in paraffin, dehydration, physical sectioning of tissue blocks, transfer to glass slides, staining, sealing, microscopy and/or digitization of sample slides using slide scanners. In the past, there have been initiatives to reduce manual hands-on-time by developing automation solutions for a number of these single steps, which however lack a high sample throughput and only match partly the efficiency of the manual sample handling. A full automation of the entire process is currently not or only to a limited degree possible as the glass slide based histology approach has intrinsic limitation in regards of preparation time, quality, completeness of the tissue sample and abilities to perform multiplex marker stainings.
In the context of detailed phenotyping of specimens and precision medicine, staining for multiple biomarkers in the identical sample becomes of high relevance. Combining this information with genetic or proteomic data from the identical sample is key to improve patient outcome. This is only possible with a tissue preserving approach which allows multiple types of analysis from the identical sample. This, in particular, has high value in low volume tissue samples, e.g. from fine needle biopsies, because the material is highly limited in volume. In addition, the current glass slide based approach results in a high burden to analyse entire tissue specimens and consequently only representative sections are analysed which results in missing spatial information aggravating the interpretation of complex phenotypes, e.g. penetration of tumours through relevant tissue architectural structures. In conclusion, advancements in pathology and histological diagnostics are limited, even though there is a high need for automation to reduce manual work since qualified staff is limited, as well as for improved and more detailed analysis of human tissue samples.
The above-mentioned points indicate that novel approaches for tissue preparation and imaging of entire tissue specimens independent from glass slides are required. Optical tissue clearing presents a suitable method for multiplex staining of entire tissue specimens. By incubating the specimen in an optical clearing reagent, the specimen becomes translucent, and therefore allows light to penetrate the tissue and consequently increase the accessible and imaging depth for optical sectioning. Available methods for optical clearing and multiplex tissue staining require expensive reagents and time-consuming incubation steps. In addition, the speed and degree of optical transparency varies depending on tissue or organ type as well as pathological entity. To increase the interpretability of such images by trained medical specialist in the field of histopathology an automated approach for optical tissue clearing with high transparency and speed is required.
US 2020/0209118 A1 disclosed the aforementioned approach for tissue preparation and imaging of entire tissue specimens. It discloses a method for preparing an animal tissue for fluorescence microscopy that allows visualization of single cells within mammalian tissues. This document discloses the technological background of visualization of single cells within tissue samples but does not disclose a histopathology analysis of biospecimens suitable for high number of tissue samples currently processed in clinical pathology laboratories.
US 10,591 ,392 B2 discloses a method of processing biopsy sized tissue samples using fluorescence microscopy. However, the disclosed method is not automated and bases on manual processing of the tissue samples.
Kewin W. Bishop, et al., ‘An end-to-end workflow for non-destructive 3D pathology’ published on August 6, 2023 under discloses an end-to-end protocol covering all aspects of a 3D pathology workflow using light sheet microscopy with sufficient detail to perform well-controlled pre-clinical and clinical studies. Guidelines for a broad range of end-users, particularly biologists, clinical researchers and engineers, are presented in a simple tutorial format.
None of the aforementioned prior art discloses an automated tissue preserving histopathologic analysis of biospecimens.
Therefore, there is a high requirement for a novel automated approach allowing automated sample processing, quality control and subsequent 3D imaging. This imaging approach should feature subcellular resolution, rapid image acquisition time, availability of multiplex marker staining, detailed spatial resolution, as well as preservation of the sample for downstream applications. The generated images have to be immediately recognizable by pathologists and microanatomists to improve speed and histopathological examination while reducing the number of skilled personnel required for sample preparation. The present invention addresses this unmet need. The object is solved according to the method disclosed in claim 1. Preferred embodiments of the invention are defined in the dependent claims.
The method for an automated tissue preserving histopathologic analysis of biospecimens according to the invention comprises at least the steps of: a) obtaining a tissue sample; b) storing the tissue sample in a tissue container, wherein the tissue container includes an identification tag; c) filling the tissue container with a dehydrating fluid for a first time-period and draining the dehydrating fluid after the first time-period; d) filling the tissue container with at least one labelling fluid for a second timeperiod and draining the at least one labelling fluid after the second time-period; e) filling the tissue container with a clearing solution; f) imaging the tissue sample in the tissue container using a 3D imaging device, particularly a light-sheet microscope; g) processing the image obtained using the 3D imaging device by: h) identifying and quantifying labelled structures, like a cell, a cellular component, cells, cell types and/or extracellular content and tissue architecture in its spatial distribution; and i) visualizing the analysed tissue sample in a 3D model and/or in multiple 2D sectional views.
The inventive method starts with receiving a tissue sample. The tissue sample is a biospecimen, which has been extracted from a human or animal, for example during a minimal-invasive surgery, an endoscopic procedure, a fine needle biopsy or similar procedure for extracting a biospecimen from a human or animal. The terms biospecimen and tissue sample can be used interchangeably. The tissue sample can consist of multiple separate biospecimen which have been obtained individually from the same human or animal and which are processed together as a single tissue sample.
According to an embodiment of the invention, the tissue sample is a fixed tissue sample. However, the step of fixing the biospecimen extracted from the human or animal body is not necessarily covered by the inventive method. The extracted biospecimen is for example fixed by treating the biospecimen with a fixing solution, particularly by immersing the biospecimen in the fixing solution. The biospecimen is kept in the fixing solution until it has been fixed. The fixing solution is for example Formalin or 4 % PFA.
Fixing the tissue sample refers to any process that halts cellular degradation and respectively a fixed tissue sample is not subject to cellular tissue degradation. In a variant of the invention, the fixed tissue sample can be washed prior to further processing with a liquid for removing the fixing solution. For example, a tissue sample fixed using formalin can be washed using a phosphate buffered saline.
Alternatively, it is also possible to process a tissue sample that is not fixed, like for example a direct (on the fly) analysis after obtaining the tissue sample from the human or animal body.
The obtained tissue sample is stored in a tissue container for further processing. The tissue container comprises an identification tag, so that the tissue container and hence the contained tissue sample, can be tracked throughout the whole process of the invention. A tracking of the tissue samples is an essential feature of the automated histopathologic analysis because the method is performed without human interaction and there must be some means for checking the status of the processing of the single tissue samples. This requires a tracking of the tissue container throughout the whole processing.
Next, the tissue container is filled with a dehydrating fluid for a first time-period and the dehydrating fluid is drained after the first time-period (referred to as step c) of the method according to the invention). The dehydrating fluid is used for dehydrating the tissue sample in the tissue container and making it miscible with the clearing fluid used during step e). Dehydrating refers to the removal of water and/or (partially) lipids from the tissue sample, which aids in preparation of the tissue sample for the later imaging of the tissue sample during step f) of the method. The dehydrating has the advantage that in the later process an anhydrous clearing reagent or a clearing fluid with low water solubility can be used during step e).
The first time-period is chosen such that the tissue sample is sufficiently dehydrated and hence depends on the size of the respective tissue sample and type of the tissue sample. Considering common tissue sample sizes between 0,1 cm and 3 cm the first time-period will be between 15 seconds and 1 hour. However, this might differ for different types of tissue samples like lung or lymph nodes.
The dehydrating fluid is preferably a water-miscible anhydrous fluid. The dehydrating fluid is chosen e.g. from methacarn or alcohols such as methanol, ethanol and propanol.
Step c) of the method can also be repeated two or more times using the same or another dehydrating fluid, particularly in different concentrations.
During the following step d) of the method according to the invention the tissue container is filled with at least one labelling fluid for a second time-period and the at least one labelling fluid is drained after the second time-period. The terms colouring liquid, labelling liquid and marking liquid can be used synonymously.
The labelling fluid is used for labelling one or more target molecules of the tissue sample contained in the tissue container. The labelling fluid is for example a fluorescent dye. In a preferred variant of the invention, the labelling fluid bases on antibodies, preferably nanobodies (VHHs, single domain antibodies), as labelling agents, wherein nanobodies are significantly smaller than antibodies and hence penetrate the tissue sample more easily. The antibodies and/or nanobodies are preferably labelled using chemical or biological compounds, like for example fluorescent dyes. If antibodies respectively nanobodies are used as labelling fluid in step d), this step d) can also be performed before step c). If the labelling fluid is a fluorescent chemical, step d) is performed after step c). A combination of both can also been performed. In that case performing step d) before step c) is preferred.
The second-time period is chosen such that the labelling fluid can penetrate the whole tissue sample contained in the tissue container and labelling agents of the labelling fluid can bind to the one or more target molecules.
During the following step e) the tissue container is filled with a clearing solution. The clearing solution is used for optically clearing the tissue sample in the tissue container. Optically clearing refers to matching the refractive index within the tissue sample, enabling (fluorescence) imaging deep within the tissue sample during the following step f) of the method according to the invention. The clearing solution is for example chosen from water-based or non-water-based solvents, e.g. inorganic solvents. In case the clearing solution is a water-based chemical, the dehydration step c) is optional and can be omitted.
Step e) of the method can also be repeated two or more times using the same or another clearing solution, particularly in different concentrations.
Step f) of the method according to the invention refers to imaging the tissue sample in the tissue container using a 3D imaging device, particularly a light-sheet microscope. However, other suitable 3D imaging devices can be used, like e.g. a handheld MRT device, Multiphoton microscope, confocal or fluorescence microscope or optical projection tomograph.
The 3D imaging provides a 3D visualization of the (internal) macro- and microstructures in the tissue sample contained in the tissue container, preferably at sub-micron to nanoscale spatial resolution. For example, a light-sheet microscope uses a thin sheet of light to excite only fluorophores within the focal volume and scans the whole tissue sample sheet by sheet (optical section by optical section). A lightsheet microscope has a true optical sectioning capability and, hence, provides sufficient axial resolution in all directions, e.g. isotropic point-spread function, restrict photobleaching and phototoxicity to a fraction of the sample and use cameras to record tens to thousands of images per second.
The image obtained using the 3D imaging device is processed in step g) of the method of the present invention. In a first step h) the labelled structures are identified and quantified. The labelled structures refer to a cell, a cellular component, cells, cell types and/or extracellular content and tissue architecture in its spatial distribution. A cellular component refers inter alia to a cell nucleus, cell nuclei, organelles, or similar. In a second step i) of the processing the analysed tissue sample is visualized in a 3D model and/or in multiple 2D sectional views.
Thus, the method results in a visualization of the tissue sample obtained during step a) in a 3D model and/or in multiple 2D sectional views. The labelled structures are shown in the visualization and a pathologist can examine the tissue sample based on the visualization.
All steps of the afore-mentioned method are executed automatically and at least steps c) to i) do not involve any human action other than optionally supervising the process. The sequence of the method steps can be changed, as far as this is technically possible. Thus, as long as steps do not sequentially depend on each other, the sequence of these method steps can be changed.
The automation of the whole process is facilitated by using a single tissue container for one specific tissue sample throughout the whole process. The processing can be supervised and controlled because of the identification tag associated with the tissue container. The identification tag is preferably an electronic tag like a RFID-tag. However, it is also possible to use optical identification tags using for example barcodes or QR-codes.
Particularly, the tissue container has a defined design and all tissue container handling devices, like one or more devices for filling the tissue container with the different fluids and the light-sheet microscope are adapted for handling this specific design of the tissue container. It is also possible to use tissue containers of different sizes, as long as all tissue containers have at least some areas in common, which are used for handling and/or processing the tissue contained respectively the tissue sample contained in the tissue container.
Preferably, the tissue container and the clearing fluid used in step e) of the method have matching refractive indices. This is particularly advantageous during imaging step f) of the inventive method.
Pursuant to a preferred embodiment of the invention, the method further comprises the step of: j) filling the tissue container with a permeabilization fluid for a third time-period and draining the permeabilization fluid after the third time-period, wherein this step is preferably performed between steps b) and c) and afterwards filing the tissue container with a blocking agent for a fourth time-period and draining the blocking agent after the fourth time-period.
The permeabilization fluid is accelerating the access of the at least one labelling fluid in step d) into the tissue sample, particularly into deeper portions of the tissue sample. This step j) is particularly useful if the at least one labelling fluid comprises antibodies because the permeabilization fluid facilitates the penetration of the antibodies into the tissue sample. If the labelling fluid comprises the smaller nanobodies the step j) is not necessarily required and can be omitted for reducing the overall process time. It is to be understood that if a permeabilization solution is used in a particular step of the methods of the invention, this does not exclude that other fluids, e.g. fluids used in other steps of the method, may also contribute to the permeabilization and improve permeabilization. For instance, a fluid for the removal of heme can be a solution that contributes to the permeabilization.
The blocking agent is for example an aqueous bovine serum albumin solution or serum of various species. The blocking agent improves the staining of sample because the blocking agent for example reduces unspecific binding of the chemical and/or labelling reagents. This increases the quality of the staining and analysis of the tissue sample.
In a variant of the invention the method comprises the step of: k) checking the clearing or transparency of the tissue sample in the tissue container after the second time-period.
This step ensures that the tissue sample in the tissue container is suitable for the imaging in step f) of the method. The checking bases for example on relative transparency compared to original tissue sample.
According to an advantageous variant of the invention, the method further comprises the step of: l) recolouring the image to match the colouring of conventional histology slides, wherein this step is preferably performed between steps h) and i).
Thus, the 3D model and/or multiple 2D sectional views created by the inventive methods have the same appearance than probes resulting from histopathology processes based on cutting and staining tissue samples and the pathologist can analyse the 3D model and/or multiple 2D sectional views as known to him, without adapting to new appearance like colouring of the tissue samples, particularly the labelled structures, like a cell, a cellular component, cells, cell types and/or extracellular content.
In a preferred variant, the method according to the invention comprises the step of: m) filling the tissue container with multiple fluorescent dyes, wherein the multiple fluorescent dyes are filled simultaneously into the tissue container or by repeating step d) multiple times. The multiple fluorescent dyes are used for labelling/staining different structures, like a cell, a cellular component, cells, cell types and/or extracellular content and tissue architecture in its spatial distribution. This can happen simultaneously, particularly if the labelling of the different structures using the multiple fluorescent dyes requires similar timing or and at least partially sequentially by repeating step d), if the labelling of the different structures using the multiple fluorescent dyes requires different timing. Thus, in the latter case the second time-period can differ for each fluorescent dye.
Pursuant to a variant of the invention, for each fluorescent dye (staining) a different channel in the 3D model or in the multiple 2D sectional views is used. Thus, the pathologist can visualize the different labelled/stained structures by enabling/disabling respective channels in the 3D model or in the multiple 2D sectional views. In this way the pathologist can visualize or suppress different labelled/stained structures during his analysis depending on the current needs.
According to a further variant of the invention, the method comprises the step of: n) decolouring the tissue sample.
This step for example comprises the steps of filling the tissue container with a decolouring fluid for a fifth time-period and draining the decolouring fluid after the fifth time-period, wherein this step is preferably performed before step d). The decolouring fluid is preferably an alcohol, hemolysis fluid, formamide, peroxide, acid and/or base, reducing and/or oxidizing agent or similar fluid. Alternatively, the tissue sample can be decoloured by a physical or biological treatment like laser treatment or enzyme treatment. The decolouring of the tissue sample enhances the penetration of light waves into the tissue sample during the imaging step f), which result in better image quality. The decolouring further enables a restaining of the tissue sample.
In an advantageous variant, the inventive method comprises the step of: o) tracking tissue container through the complete process, particularly based on the identification tag of the tissue container.
In an automated process it is useful to continuously track the progress of the process, i.e. where are different tissue containers, what is the current process step for each tissue container, how long until the results are available for each tissue sample, and so on. The basis for this information is the continuously tracking of the tissue container through the whole process. The tracking preferably bases on the identification tag of the tissue container.
Pursuant to a variant of the invention, the method comprises the additional step of: p) rehydrating the tissue sample in the tissue container.
After rehydrating the tissue sample in the tissue container, the tissue sample can be processed by downstream applications such as regular histology, protein applications, archiving and/or DNA/RNA applications. The method according to the invention is tissue preserving and hence the tissue sample, although already analysed by the inventive method, can be analysed by regular histology and/or DNA/RNA applications. However, in this case the tissue sample is preferably rehydrated.
According to an expedient variant of the invention, the method comprises the step of: q) removing the one or more staining from the tissue sample, which have been caused by the one or more fluorescent dyes.
Thereby the tissue is transferred back to its original state as it would not have been stained before and hence can be further processed.
In a variant of the invention, the one or more staining is removed from the tissue sample by biological, physical and/or chemical treatment, particularly using a destaining fluid or solution. The destaining fluid or solution is preferably an alcohol, formamide, peroxide, acid and/or base, reducing and/or oxidizing agent or similar fluid. Alternatively, the tissue sample can be destained by a physical or biological treatment like laser treatment.
Pursuant to a preferred variant of the invention, the method comprises the further step of: r) controlling the automated tissue preserving histopathologic analysis of biospecimens using a central process control, wherein the central process control comprises one or more of the following information: defining kind of tissue sample, origin of tissue sample, relevant analysis to be performed, required stainings, defining time-periods for different steps of method, tracking and/or recording of process steps and the corresponding results, or similar variables and/or features of the histopathologic analysis. The central process control is for example a computer system having communication interfaces to devices executing the different step of the inventive method, like a device for filling and draining one or more fluids to tissue containers, the imaging device like a light-sheet microscope, devices for handling, particularly moving, the tissue containers or similar devices. The central process control preferably uses a graphical user interface for user interaction. Furthermore, the central process control can display or forward the result of the imaging process, particularly for a pathologist for analysing the imaging result.
According to an advantageous variant of the invention, the method comprises the step of: s) agitating the tissue container during step c), d), e), j), m) and/or n).
Agitating the tissue container during step c), d), e), j), m) and/or n) improves the penetration of the respective fluid filled into the tissue container during the corresponding step with the tissue sample contained in the tissue container. This reduces the respective time-period required for treating the tissue sample with the respective fluid. Thus, the agitation of the tissue container reduces the overall process time, which has a great impact during automatic mass-analysis of thousands of tissue samples, which are currently analysed manually in glass slide based histology approach.
In a preferred variant of the invention, the method is performed at room temperature. Thus, the method does not involve any heating of the tissue container, respectively the content of the tissue container during any of the method steps. Room temperature according to the present invention refers to a common temperature in a laboratory, e.g. between 15°C and 25°C.
Pursuant to an advantageous variant of the invention, the method comprises the further step of: t) checking characteristics of the fluid used during step c), d), e), j), m) and/or n) and comparing the result to the expected characteristics of the fluid used in the respective step.
This step is an in-process control of the liquid filled into the tissue container during the respective steps. Any of the liquid has a defined characteristic and hence, it can be checked whether the liquid container has been filled with the correct liquid. This is particularly useful, if a single device is used to fill and drain different liquids to the tissue container.
According to a further expedient variant of the invention, the method comprises the step of: u) checking the fill level of the tissue container during step c), d), e), j), m) and/or n).
Thus, it is checked whether the tissue container has been filled as defined, i.e. that the tissue sample inside the tissue container is completely surrounded by the liquid filled into the tissue container. This guarantees that the tissue sample is effectively and completely treated by the liquid filled into the tissue container.
Pursuant to a further variant of the invention, the fill level of one or more containers containing the one or more fluids used during steps c), d), e), j), m) and/or n) may be checked respectively monitored. Thereby it is guaranteed that sufficient amounts of these fluids are available during performing the inventive method.
In a variant of the invention, the tissue container has a tissue sample chamber having a volume of 1000 mm3 to 3000 mm3, preferably about 2000 mm3. For example, the tissue sample chamber is cylindrically, having a base area of 79 mm2 and a height of 25 mm.
In the following the invention will be further explained with respect to embodiments shown in the figures. It shows:
Fig. 1 a flow diagram of a method for an automated tissue preserving histopathologic analysis of biospecimens according to the present invention;
Fig. 2 a tissue sample before step c) and after step e) of the method according to the present invention; and
Fig. 3 the visualization process of a tissue sample according to the present invention.
Fig. 1 shows a flow diagram of a method for an automated tissue preserving histopathologic analysis of biospecimens according to the present invention. The inventive method starts with receiving a tissue sample, also referred to as step a) of the method according to the invention. This tissue sample is a biospecimen, which has been extracted from a human or animal, for example during a minimal-invasive surgery, an endoscopic procedure, a needle biopsy or similar procedure for extracting a biospecimen from a human or animal. According to the embodiment of Fig. 1 the tissue sample is a fixed tissue sample. However, the step of fixing the biospecimen extracted from the human or animal body is not necessarily covered by the inventive method. The extracted biospecimen is for example fixed by treating the biospecimen with a fixing solution, particularly by immersing the biospecimen in the fixing solution. The biospecimen is kept in the fixing solution until it has been fixed. The fixing solution is for example Formalin or 4 % PFA.
In the next step, also referred to as step b), the obtained tissue sample is stored in a tissue container for further processing. The tissue container comprises an identification tag, so that the tissue container and hence the contained tissue sample, can be tracked throughout the whole process of the invention. The tracking of the tissue samples is an essential feature of the automated histopathologic analysis because the method is performed without human interaction and there must be some means for checking the status of the processing of the single tissue samples.
In the next sequence of steps the tissue sample in the tissue container is prepared for 3D imaging. These sequence of steps comprises the single steps of dehydrating (step c)), labelling (step d)) and clearing (step e)) the tissue sample in the tissue container.
During step c) the tissue container is filled with a dehydrating fluid for a first time-period and the dehydrating fluid is drained after the first time-period. The dehydrating fluid is used for dehydrating the tissue sample in the tissue container and making it miscible with the clearing fluid used during step e). Dehydrating refers to the removal of water and/or (partially) lipids from the tissue sample, which aids in preparation of the tissue sample for the later imaging of the tissue sample during step f) of the method. The dehydrating has the advantage that in the later process an optical clearing fluid with low water solubility can be used during step e). The dehydrating fluid is a water- miscible anhydrous fluid. The dehydrating fluid is chosen e.g. from methacarn or alcohols such as methanol, ethanol and propanol.
During step d) of the method according to the invention the tissue container is filled with at least one labelling fluid for a second time-period and the at least one labelling fluid is drained after the second time-period. The labelling fluid is used for labelling one or more target molecules of the tissue sample contained in the tissue container. The labelling fluid is for example a fluorescent dye. In a preferred variant of the invention, the labelling fluid bases on antibodies, preferably nanobodies, as labelling agents. The antibodies and/or nanobodies are preferably labelled using chemical or biological compounds, like for example fluorescent dyes. If antibodies respectively nanobodies are used as labelling fluid in step d), this step d) can also be performed before step c). If the labelling fluid is a fluorescent chemical, step d) is performed after step c). In case of combining both antibodies I nanobodies and fluorescent chemical, step d) is performed before step c).
During the step e) the tissue container is filled with a clearing solution, like for example chosen from water or water-based inorganic solvents. The clearing solution is used for optically clearing the tissue sample in the tissue container. Optically clearing refers to matching the refractive index within the tissue sample, enabling fluorescence imaging deep within the tissue sample during the following step f) of the method according to the invention.
The next step f) of the method according to the invention refers to imaging the tissue sample in the tissue container using a 3D imaging device, particularly a light-sheet microscope. However, other suitable 3D imaging devices can be used, like e.g. a handheld MRT device, Multiphoton microscope, confocal or fluorescence microscope or optical projection tomograph.
The 3D imaging provides a 3D visualization of the internal microstructures in the tissue sample contained in the tissue container, preferably at sub-micron to nanoscale spatial resolution.
In step g) of the method according to the invention the images obtained during step f) are processed. In a first step h) of the processing the labelled structures are identified and quantified. The labelled structures refer to a cell, a cellular component, cells, cell types and/or extracellular content and tissue architecture in its spatial distribution. A cellular component refers inter alia to a cell nucleus, cell nuclei, organelles, or similar. In a second step i) of the processing the analysed tissue sample is visualized in a 3D model and/or in multiple 2D sectional views. Thus, the method results in a visualization of the tissue sample obtained during step a) in a 3D model and/or in multiple 2D sectional views. The labelled structures are shown in the visualization and a pathologist can examine the tissue sample based on the visualization.
All steps of the method are executed automatically and at least steps c) to i) do not involve any human action other than optionally supervising the process.
Fig. 2 shows a tissue sample before step c) and after step e) of the method according to the present invention. Thus, Fig. 2 shows the obtained tissue sample before and after preparing the tissue sample during steps c), d) and e). Thus, Fig. 2 visualizes the preparation of the tissue sample for the imaging.
Fig. 3 shows the visualization process of a tissue sample according to the present invention. The images obtained e.g. by a light-sheet microscope are for example processed sheet by sheet, i.e. the input is 2D image (sheet). The sheets obtained during imaging are processed individually and at the end are visualized together in a 3D visualization of an image stack.

Claims

1. Method for an automated tissue preserving histopathologic analysis of biospecimens comprising the steps of: a) obtaining a tissue sample; b) storing the tissue sample in a tissue container, wherein the tissue container includes an identification tag; c) filling the tissue container with a dehydrating fluid for a first time-period and draining the dehydrating fluid after the first time-period; d) filling the tissue container with at least one labelling fluid for a second time-period and draining the at least one labelling fluid after the second time-period; e) filling the tissue container with a clearing solution; f) imaging the tissue sample in the tissue container using a 3D imaging device, particularly a light-sheet microscope; and g) processing the image obtained using the 3D imaging device by: h) identifying and quantifying labelled structures, like a cell, a cellular component, cells, cell types and/or extracellular content and tissue architecture in its spatial distribution; and i) visualizing the analysed tissue sample in a 3D model and/or in multiple 2D sectional views.
2. Method for the automated tissue preserving histopathologic analysis of biospecimens according to claim 1 , further comprising the step of: j) filling the tissue container with a permeabilization fluid for a third timeperiod and draining the permeabilization fluid after the third time-period, wherein this step is preferably performed between steps b) and c) and afterwards filing the tissue container with a blocking agent for a fourth time-period and draining the blocking agent after the fourth time-period.
3. Method for the automated tissue preserving histopathologic analysis of biospecimens according to claim 1 or claim 2, further comprising the step of: k) checking the clearing or transparency of the tissue sample in the tissue container after the second time-period.
4. Method for the automated tissue preserving histopathologic analysis of biospecimens according to any of claims 1 to 3, comprising the step of: l) recolouring the image to match the colouring of conventional histology slides, wherein this step is preferably performed between steps h) and i).
5. Method for the automated tissue preserving histopathologic analysis of biospecimens according to any of claims 1 to 4, comprising the step of: m) filling the tissue container with multiple (fluorescent) dyes, wherein the multiple (fluorescent) dyes are filled simultaneously into the tissue container or by repeating step d) multiple times.
6. Method for the automated tissue preserving histopathologic analysis of biospecimens according to claim 5, wherein for each (fluorescent) dye, respectively staining, a different channel in the 3D model or in the multiple 2D sectional views is used.
7. Method for the automated tissue preserving histopathologic analysis of biospecimens according to any of claims 1 to 6, comprising the step of: n) decolouring the tissue sample.
8. Method for the automated tissue preserving histopathologic analysis of biospecimens according to any of claims 1 to 7, comprising the step of: o) tracking tissue container through the complete process, particularly based on the identification tag of the tissue container.
9. Method for the automated tissue preserving histopathologic analysis of biospecimens according to any of claims 1 to 8, comprising the step of: p) rehydrating the tissue sample in the tissue container.
10. Method for the automated tissue preserving histopathologic analysis of biospecimens according to any of claims 1 to 9, comprising the step of: q) removing the one or more staining from the tissue sample, which have been caused by the one or more (fluorescent) dyes.
11. Method for the automated tissue preserving histopathologic analysis of biospecimens according to claim 10, wherein the one or more staining is removed from the tissue sample preferably by photocleaving or destaining fluid or solution like ethanol.
12. Method for the automated tissue preserving histopathologic analysis of biospecimens according to any of claims 1 to 11 , further comprising the step of: r) controlling the automated tissue preserving histopathologic analysis of biospecimens using a central process control, wherein the central process control comprises one or more of the following information: defining kind of tissue sample, origin of tissue sample, relevant analysis to be performed, required stainings, defining time-periods for different steps of method, tracking and/or recording of process steps and the corresponding results.
13. Method for the automated tissue preserving histopathologic analysis of biospecimens according to any of claims 1 to 12, further comprising the step of: s) agitating the tissue container during step c), d), e), j), m) and/or n).
14. Method for the automated tissue preserving histopathologic analysis of biospecimens according to any of claims 1 to 13, wherein the method is performed at room temperature.
15. Method for the automated tissue preserving histopathologic analysis of biospecimens according to any of claims 1 to 14, further comprising the step of: t) checking characteristics of the fluid used during step c), d), e), j), m) and/or n) and comparing the result to the expected characteristics of the fluid used in the respective step.
16. Method for the automated tissue preserving histopathologic analysis of biospecimens according to any of claims 1 to 15, further comprising the step of: u) checking the fill level of the tissue container during step c), d), e), j), m) and/or n).
17. Method for the automated tissue preserving histopathologic analysis of biospecimens according to any of claims 1 to 15, wherein the tissue sample is a fixed tissue sample.
EP24837564.4A 2023-12-20 2024-12-19 Method for an automated tissue preserving histopathologic analysis of biospecimens Pending EP4630780A1 (en)

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