Sample preparation method for microscopy using chitosan-based porous materials

Abstract

The present invention relates to a method for preparing samples for microscopy, comprising the steps of: using a chitosan-based porous material having interconnected pores with a size between 5 μm and 700 μm and a total porosity (volume fraction) between 40% and 90%; adding at least one biopsy sample containing tissue and / or eukaryotic and / or prokaryotic cells and / or eukaryotic cells containing viral particles to said chitosan-based porous material; and (C 1 -C 6 )-alkyl alcohol or mixtures thereof.

Description

[Technical field]

[0001] The present invention is directed to a method of preparing samples for microscopic examination, and in particular, to the pathological evaluation of fresh biopsy samples using chitosan-based porous materials. [Background technology]

[0002] The use of chitosan-based materials for the encapsulation of cell preparations has also been described in WO2018 / 083616A1 and in the article "CytoMatrix for a reliable and simple characterization of lung cancer stem cells from malignant pleural effusions" by Bruschini, S. et al. in J. Cell Physiol. 2020;235;1877-1887. In this article, the use of a chitosan-based material named "CytoMatrix" as a support for the characterization of limited amounts of cancer cells isolated from malignant pleural effusions (MPEs) of patients suffering from non-small cell lung cancer is described. The cell samples are loaded onto CytoMatrix™ cell blocks, followed by formalin fixation and paraffin embedding. The cell blocks are then cut into 5 μm sections using a microtome, and these sections are mounted on slides for microscopic evaluation.

[0003] A typical histopathological workflow involves fixation in formalin for several hours, embedding the biopsy in so-called "embedding resins" such as Cryomatrix™ or paraffin, cutting the embedded blocks into sections, mounting the sections on microscope slides, followed by staining, typically with hematoxylin-eosin stain, for example. These steps are time-consuming, labor-intensive, require bulky laboratory equipment, and are generally difficult to set up and synchronize with the surgical workflow. In such a histopathological workflow, the sections may be destroyed (e.g., when cutting the sections) and thus wasted, and thus the diagnostic section information is lost.

[0004] In WO / 2018 / 083616A1 a novel chitosan-based material is described, which is today commercially available under the trade name CytoMatrix™, which is useful for histopathological sample preparation. According to the workflow disclosed in the CytoMatrix™ data sheet, the sample is immersed in formalin for at least 6-8 hours if the biopsy sample is pretreated before loading on CytoMatrix™, or for at least 12 hours if the sample is applied directly on CytoMatrix™ (see https: / / cytomatrix.it). This means that the results of the biopsy are only available the day after the biopsy, and medical personnel, e.g. surgeons, can only know the diagnostic result or the success of the biopsy procedure at a much later stage.

[0005] It is therefore desirable, particularly for medical personnel and patients, to obtain results from a biopsy shortly thereafter, and preferably during the biopsy procedure.

[0006] The inventors have now discovered that it is possible to use the chitosan-based porous materials described in WO2018 / 083616A1, in particular the material available under the trade name CytoMatrix™, for preparation of samples for microscopy without time-consuming fixation of the biopsy sample through immersion in formalin for several hours.

[0007] Thus, the present invention relates to a novel sample preparation procedure for rapid microscopic evaluation of pathological biopsies, which are evaluated within a short time frame, preferably during a biopsy procedure, which may be a biopsy session or a surgical procedure, e.g., removal of malignant (e.g. cancerous or infected) tissue.

[0008] This is advantageous because the patient remains present and the desired next steps (e.g., surgery or repeat biopsy) can be immediately determined by medical personnel based on the evaluation of the biopsy results, and in particular the information provided by the microscopic images.

[0009] This new rapid pathological evaluation method according to the present invention offers many benefits for medical professionals (e.g. surgeons, endoscopists, operating / hospital staff) and for the patient in terms of cost, potential pain and suffering, and also reduces labor time as it allows the histopathologist to evaluate the biopsy sample immediately after and during the biopsy procedure.

[0010] Examination of the biopsy sample is performed on optical image sections, not on cut sections. Optical image sections are images from thin sections of the biopsy sample. They are acquired using an appropriate microscopic imaging technique, such as laser scanning confocal technology. By using optical image sectioning, the biopsy remains intact, as opposed to using cut sections prepared according to a conventional histopathological workflow. In a standard histopathological workflow, some sample sections are destroyed and wasted during cutting, and thus section information for diagnosis is lost. Optical image sectioning also takes less time, is less labor intensive, and does not require bulky laboratory equipment. In addition, biopsy sample preparation and its examination can be synchronized with the surgical workflow.

[0011] Furthermore, the location for evaluation of microscopy data, for example in the form of digital microscopy images, may be on-site or off-site, i.e. near where the biopsy was taken or may be shared in real time with external experts.

[0012] The histopathological findings can guide the surgeon through the operation. Based on the findings, the surgeon can make immediate treatment decisions as early as in the operating room, if necessary. For example, if the biopsy is positive for cancer cells, the surgeon removes the tumor or even the tumor border, so that the patient does not have to go through the same procedure again later. Thus, the immediate evaluation of the biopsy sample helps the surgeon to remove all border tissue and eliminate the possibility of tumor recurrence. Alternatively, if the biopsy is negative for cancer cells, the surgeon does not unintentionally remove more healthy tissue than necessary. This is beneficial for the patient, as it can avoid the negative physiological and psychological side effects of removing healthy tissue, and the patient's appearance (e.g., face or body shape) is not changed excessively. Also, if no cancer cells are observed in the fine needle aspiration biopsy of the lymph node, the removal of the lymph node during cancer surgery can be omitted. In breast cancer surgery, the axillary nodes are removed as a standard preventative measure.

[0013] Generally, if a repeat biopsy is required for the reasons mentioned above or because the biopsy is of poor quality and quantity to make a diagnosis, medical personnel and the patient can still be on-site and perform the repeat biopsy immediately.

[0014] In addition to the simplicity of sample preparation, another advantage of the present invention is that it is possible to use a benchtop microscope (such as the VivaScope™ 2500) and therefore examination of the biopsy sample can be performed under sterile conditions, i.e., in an operating room or a sterile operating section of a hospital.

[0015] Therefore, in [embodiment 1], the present invention relates to a method for preparing a sample for microscopy, comprising the following steps: Step (i): using a solid or ductile chitosan-based porous material having interconnected pores with a size between 5 μm and 700 μm and a total porosity (volume fraction) between 40% and 90%; Step (ii): Adding at least one biopsy sample containing tissue and / or eukaryotic and / or prokaryotic cells and / or eukaryotic cells containing viral particles to said chitosan-based porous material; and Step (iii): Adding a (C1-C6)-alkyl alcohol or a mixture thereof The present invention is directed to a method comprising:

[0016] In [Embodiment 2], the present invention is directed to the method described in [Embodiment 1], in which the chitosan-based porous material is obtained by preparing a chitosan and / or lactosylated chitosan solution or a vinyl derivative solution of chitosan, the vinyl derivative being alone or mixed with a sulfhydryl derivative of chitosan, all compounds having a molecular weight between 50 kDa and 200 kDa, dissolved in an acid solution of a polar inorganic or organic acid, followed by gelation of the solution to obtain a hydrogel, and freeze-drying the resulting hydrogel to obtain the porous material.

[0017] In [Embodiment 3], the present invention is directed to the method of [Embodiment 1] or [Embodiment 2], wherein a (C1-C6)-alkyl alcohol or a mixture thereof is used in step (iii), and the method contains at least 5% water or an aqueous buffer solution.

[0018] In [embodiment 4], the present invention is directed to a method according to any one of the previous embodiments, further comprising step (iv), i.e., staining the biopsy sample by adding at least one fluorescent dye, the dye being preferably selected from 2,3-benzoquinoline type dyes, acridine orange, acriflavine, proflavine and acridine yellow, thiazine type dyes, toluidine blue and methylene blue, Masson's trichrome staining colour, methyl blue, aniline blue, fast green FCF, water blue, hematoxylin, eosin, ethidium bromide, rhodamine 123, cyber green, acid fusion, sirius red, Col-F, Nile blue, Nile red, oil red O, auramine O, neutral red, patent blue, fluorescein, indocyanine green (ICG), methyl green, pyronine Y and combinations thereof. Among the above mentioned dyes, 2,3-benzoquinoline dyes, in particular acridine orange, are preferably used. The dye may contain a linker molecule, such as an antibody or a lectin. When the dye is bound to the linker molecule, the linker interacts with cells and / or tissues of the biological sample.

[0019] In [Embodiment 5], the present invention is directed to a method according to any one of [Embodiment 1] to [Embodiment 4], wherein the chitosan-based porous material is used in the form of a slab or block, and at least one surface of the slab or block is capable of receiving the biopsy sample as a whole when collected via incisional biopsy, stereotactic biopsy, sternal biopsy, chorionic villus biopsy, cone biopsy, endoscopic biopsy, needle biopsy (percutaneous biopsy), fine needle aspiration biopsy (FNA), fine needle biopsy (FNB), small needle biopsy, microneedle biopsy, core needle biopsy or cell suspension biopsy.

[0020] The shape and size (width, length and height) of the chitosan-based porous material are selected so as to meet the requirements of the specimen holder of the microscope used for pathological examination, especially for optical image sectioning, and to be able to receive as many biopsy samples as possible, especially without the need to fragment the sample.Using the method according to the invention, cutting the chitosan-based porous material into sections after the sample is mounted on the support and before microscopic examination can be omitted, thus again simplifying the sample preparation compared to standard histopathological preparation methods using formalin-fixed sections for microscopic evaluation.

[0021] In one embodiment of the invention, the length and / or width, or diameter, of the at least one surface of the slab or block capable of receiving the biopsy sample is adapted to the type of biopsy sample for which it is used and to the size of the sample holder of the microscope used. The appropriate size can be easily determined by a person skilled in the art. For example, if a microneedle biopsy is examined with the method according to the invention using a microscope of the VivaScope™ 2500 type, the length of one side of the slab or block is preferably at least 0.8 cm.

[0022] When using confocal microscopy, particularly a microscope of the VivaScope™ 2500 type, the size of the block or slab depends on the stiffness of the chitosan-based porous material, and in the context of [embodiment 2] or CytoMatrix™, the size preferably ranges from 0.8 cm x 0.8 cm x 0.2 cm to 1.5 cm x 1.5 cm x 0.2 cm in size, including sizes such as 0.8 cm x 1.5 cm (width x length). Smaller and larger sizes are possible.

[0023] The VivaScope™ 2500 microscope uses its laser scanning confocal technology to provide high quality optical section images at cellular resolution. It provides optical laser scanning sections without the time-consuming and labor-intensive microtome sectioning of conventional standard cytopathology or histopathology procedures, and rapidly images samples within minutes. In addition, the digitized images can be shared remotely with histopathologists in real time.

[0024] The adaptation of the CytoMatrix™ size from the current commercially available block size of 0.8 cm x 0.8 cm to the sizes mentioned herein allows more biopsy samples to be loaded onto the same CytoMatrix™, thus allowing the imaging of several biopsies in only one imaging session. Since biopsy specialists or surgeons usually take several biopsies in one session, the larger the size chosen, the less the overall microscopy imaging time will be in favor of a faster diagnosis.

[0025] In embodiment 6, the chitosan-based porous material used in step (i) of the method of the present invention is used in an open-top lattice box with at least one compartment adapted to the desired size of the slab or block.The use of chitosan-based porous material in such a box is advantageous to facilitate the handling of the chitosan-based porous material, which is often elastic.

[0026] In [Embodiment 7], a sample prepared according to any one of the preceding embodiments is mounted on a specimen or sample holder for use in microscopy for examination of the biopsy sample, representing step (v) of the method of the present invention.

[0027] A suitable specimen holder comprises a holder in which the specimen is clamped between a first plate and a second plate with a window through which the optical beam passes. For fastening the first and second plates, the plates may comprise magnetic means so that the first and second plates are magnetically attracted. Such specimen holders are known and are described in DE102019101035A1. For the accuracy of the microscopic evaluation, it is important that the specimen is securely fastened on the microscope specimen holder during acquisition of the microscope data of the optical image section. Otherwise, if the microscope specimen starts to move during data collection (independently of the microscope stage movement), scanning of the optical image section is not possible.

[0028] The inventors have found it advantageous to use a magnetic specimen holder as described in DE102019101035A1, but modified in that the elastic cushion is omitted and a spacer is used. The spacer is placed between the magnets of the lower and upper plates without or only partially modulating the magnetic attraction between the magnets of the lower and upper plates. The use of a spacer is necessary because the magnetic pressure compresses the microscope specimen and may damage it. Such a specimen holder is illustrated in figures 3 and 4. The invention therefore also relates to the preparation of a specimen for microscopy as described herein, in particular in the above-mentioned embodiment, using a magnetic specimen holder as shown in figures 3 and 4, which includes a spacer placed between the magnetic closures of the upper and lower plates of the specimen holder. The invention also relates to a magnetic specimen holder as described herein.

[0029] In addition to the advantages mentioned above, the mounting of the microscope sample on the magnetic holder of the present invention ensures the flatness of the imaging surface and its correct localization within the focal length of the objective lens, which is crucial to enable focused image acquisition of the biopsy sample, i.e. optical image sectioning, and thus diagnosis.

[0030] The spacer material may be inorganic or organic, natural or synthetic elastomers, plastomers or any type of dough or plaything.

[0031] The invention is further directed to a kit comprising a sample prepared by the method of the invention, and a magnetic specimen holder comprising a chitosan-based material and a spacer.

[0032] Moreover, the present invention is directed to the use of a sample prepared by the method according to the present invention, particularly as described in the above embodiments, for immediate (instant) testing of the biopsy sample after collection. Immediately means that a fresh biopsy sample can be imaged as soon as it is taken, without performing a lengthy fixation step using formalin for at least 6 hours. The time frame for a biopsy sample to be considered "fresh" typically ranges from immediately after biopsy to the point at which the sample begins to deteriorate without the application of a preservation method to the sample.

[0033] The inventors have found that step (iii), i.e. the addition of at least one (C1-C6)-alkyl alcohol, enhances the penetration of the fluorescent dye into the biopsy sample, which is also believed to partially fix the biopsy to the chitosan-based porous material. The (C1-C6)-alkyl alcohols used are, for example, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-1-propanol, 2-methyl-2-propanol, cyclohexanol, preferably ethanol. The concentration of the alcohol used is preferably in the range of 50% (v / v) to 95% (v / v), preferably in the range of 65% (v / v) to 75% (v / v). The alcohol may be brought to the desired concentration by adding water or a suitable aqueous buffer. A suitable buffer is one that has a stabilizing effect on the cells contained in the biopsy sample. The skilled person knows how to select a suitable buffer in view of the biopsy sample to be examined.

[0034] The inventors have found that ethanol is particularly suitable in this respect, especially when used at a concentration of 65% (v / v) to 75% (v / v), as it provides a good permeabilization effect without damaging the cell morphology of the biopsy sample. The use of 70% (v / v) ethanol is particularly advantageous.

[0035] Alcohol is used in an appropriate amount, which may range from 50 μL to 2 mL. This depends essentially on the size of the chitosan-based porous material used and on the volume of the biopsy sample. If a microscope of the type VivaScope™ 2500 is used for microscopic evaluation, 50 μL to 500 μL is appropriate depending on the volume of the biopsy to be examined. Alcohol enhances the permeabilization of the biopsy sample, thus causing rapid and efficient penetration of the fluorescent dye into the biopsy during staining, often within just a few seconds, and promoting uniform staining throughout the biopsy sample. Therefore, the alcohol treatment ensures uniform staining of the biopsy sample, which is necessary for evaluation / diagnosis. Without the effects described above (e.g., the penetration of the fluorescent dye into the biopsy enabled by alcohol), some of the detailed cell morphology, which is necessary for accurate biopsy evaluation, cannot be visualized in the resulting image. The beneficial effect of the addition of alcohol is illustrated in Figures 2a / 2b and 2c / 2d.

[0036] The amount of dye to be added in step (iv) depends on the type of dye and the biopsy sample, in particular the type of biological material to be examined, and can be found by the skilled artisan through standard experimentation or is described by the dye manufacturer. After addition of the dyes, either alone or as a combination of dyes, and after a short incubation period, excess dye is removed by rinsing the sample with a suitable, preferably aqueous and buffered, solution.

[0037] In step (iv) of the method according to the invention, all suitable dyes capable of visualising cells and / or tissues can be used and can be used together with microscopy methods. Preferably, fluorescent dyes are used, alone or in combination with other (fluorescent) dyes. Fluorescent dyes are preferred. The dyes can interact with the cells and / or tissues of the sample directly or indirectly via linker molecules capable of binding the cells and / or tissues, such as for example antibodies and lectins.

[0038] The following dyes, which are commonly used to stain / stain biopsy samples, may be mentioned as examples of suitable dyes: 2,3-Benzoquinoline type dyes, such as acridine orange, acriflavine, proflavine and acridine yellow and their derivatives. Thiazine type dyes, such as toluidine blue and methylene blue and their derivatives. Masson's trichrome staining colors, methyl blue, aniline blue, fast green FCF, water blue, hematoxylin, eosin and their derivatives. Ethidium bromide, rhodamine 123, Cyber ​​Green, Acid Fusion, Sirius Red, Col-F, Nile Blue, Nile Red, Oil Red O, Auramine O, Neutral Red, Patent Blue, Fluorescein, Indocyanine Green (ICG), Methyl Green, Pyronine Y, and their derivatives.

[0039] They can be used alone or as a mixture of at least two dyes.

[0040] As mentioned above, suitable chitosan-based porous materials are prepared according to the method described in WO2018 / 083619A1 to obtain a material with interconnected pores with a size between 5 μm and 700 μm and a total porosity (volume fraction) between 40% and 90%. For the determination of pore size, a scanning electron microscope (SEM) can be used. The same applies to the determination of the total porosity. The material can be obtained by preparing a solution of chitosan and / or lactosylated chitosan or a solution of vinyl derivatives of chitosan, the vinyl derivatives being alone or mixed with sulfhydryl derivatives of chitosan, all compounds having a molecular weight between 50 kDa and 200 kDa, dissolved in an acid solution of a polar inorganic or organic acid, followed by gelation of the solution to obtain a hydrogel, freeze-drying the resulting hydrogel, and obtaining the porous material.

[0041] The chitosan-based porous material renders the biopsy sample intact so that it can be handled, and the use of this material protects the biopsy sample from being lost or damaged during further processing, i.e. staining and imaging, after which storage of the sample and, if desired, further pathological analysis, such as immunostaining or DNA analysis, can be performed.

[0042] A biopsy sample is a biological sample containing tissue and / or eukaryotic and / or prokaryotic cells and / or eukaryotic cells containing virus particles. Samples can be obtained from cell-containing body fluids, such as blood, urine, sputum, exudates, lavage fluids, saliva and other aqueous body fluids, or by performing biopsies, such as incisional biopsy, cervical biopsy, shave biopsy, brush biopsy (e.g., skin cytological samples), swab biopsy, stereotactic biopsy, sternal biopsy, chorionic villus biopsy, cone biopsy, endoscopic biopsy, adhesive patch-based biopsy, and the like, as well as from needle biopsies (percutaneous biopsies), such as fine needle aspiration biopsy (FNA), fine needle biopsy (FNB), small needle biopsy, micro-core needle biopsy and core needle biopsy. Biopsy samples obtained through needle biopsies, such as fine needle aspiration biopsy, small needle biopsy, fine needle biopsy and micro-core needle biopsy, in particular through FNA and FNB, are preferably used in the method according to the invention.

[0043] FNA and FNB are known biopsy methods (see DOOLEY, William C. Biopsy Techniques in Non-palpable or Palpable Breast Lesions. In: Breast Disease. Springer, Cham, 2016. S. 3-11, or TIAN, Li, et al. Evaluation of 22G fine-needle aspiration (FNA) versus fine-needle biopsy (FNB) for endoscopic ultrasound-guided sampling of pancreatic lesions: a prospective comparison study. Surgical endoscopy, 2018, 32. Jg., Nr. 8, S. 3533-3539).

[0044] While biopsy samples obtained via needle biopsy and brush biopsy can be placed directly on chitosan-based porous materials, it is often advantageous to concentrate the cells of the body fluid before adding it to the porous material. Concentration of the sample is preferably performed by centrifugation. The resulting cell-containing pellet can be resuspended in an appropriate amount of a suitable solution, preferably a cell protection buffer. Also, the retrieved biological material obtained from adhesive patch-based biopsy or brush biopsy needs to be pretreated, for example by using a cell protection buffer, before being added onto the chitosan-based porous material in that the cells are removed from the patch or brush. The cells in the resulting sample solution can be concentrated by centrifugation and loaded onto the chitosan-based porous material as described above.

[0045] Biopsy samples, especially those obtained from fine needle aspiration biopsies, small needle biopsies, fine needle biopsies, and microcore needle biopsies, are often fragile, brittle, and lacking integrity. They cannot be properly handled according to standard cytological sample preparation procedures. Handling often results in at least partial loss of the sample, and even worse, cell damage. Thus, the results of the test may be incomplete, at least partially erroneous, and therefore misleading.

[0046] After carrying out the method of the present invention, the sample containing the chitosan-based material and the biopsy sample is preserved and treated according to standard histological sample preparation, for example as described in WO2018 / 083616A1.

[0047] All available microscopes can be used for the examination of the samples prepared by the method according to the invention, preferably microscopes capable of generating optical image sections as explained above. Preferably, a laser scanning confocal microscope is used. Such microscopes are, for example, those of the VivaScope™ series sold by MAVIG GmbH (Germany) or the HistologScanner™ sold by Saman Tree Medical SA (Switzerland).

[0048] Other microscopes capable of generating optical image sections are also suitable, such as, for example: Multiphoton fluorescence microscope, Two-photon or three-photon fluorescence microscopes, for example the FD1070 sold by Femti Diagnostics BV (The Netherlands); Optical coherence tomography, marketed, for example, by Perimeter Medical Imaging, Toronto, Canada; Full-field optical coherence tomography, e.g. the Light-CT Scanner from LLTech. Microscopy using ultraviolet sectioning excitation, for example as sold by Muse Microscopy Inc., Irvine, Calif. (USA); High and ultra-high resolution subsurface imaging, Stimulated Raman scattering, For example, stimulated Raman diffusion tissue imaging sold by InVenioImaging Inc., Santa Clara, Calif. (USA); Multi-harmonic generation microscopy, second harmonic generation (SHG) or third harmonic generation (THG).

[0049] The invention is illustrated in the figures and experiments described in more detail below, without restricting or limiting the application to these figures or examples. [Brief description of the drawings]

[0050] [Figure 1a] FIG. 1 illustrates the loading of a fine needle aspiration biopsy 101 onto a chitosan-based material 100 with a syringe used for FNA.

[0051] [Figure 1b] Photograph in plan view of an FNA biopsy 101 on a chitosan-based material 100 after alcohol treatment and staining with acridine orange, but before image acquisition. The photograph has been retouched by inverting the colors. The dotted lines delineate the borders of the loaded biopsy.

[0052] [Figure 1c] FIG. 1 illustrates the loading of a fine needle biopsy 103 having a tubular shape / structure onto a chitosan-based material 100.

[0053] [Figure 1d]Photograph in plan view of fine needle biopsy 101 on chitosan-based material 100 after alcohol treatment and staining with acridine orange, but before image acquisition. The photograph has been modified by inverting the colors. Tubular fragments of tissue from FNB are prone to breakage. The chitosan material supports the FNB structure and protects it from damage or loss.

[0054] [Figure 2a] The top photograph shows a microscopic image of an FNA biopsy mounted on a chitosan-based material stained with acridine orange but without alcohol treatment. The bottom photograph is a magnified and higher powered version of the indicated section within the rectangle. The morphology of the sample is barely discernible.

[0055] [Figure 2b] The top photograph shows a microscopic image of the same FNA biopsy shown in FIG. 2a on a chitosan-based material that has been re-stained and treated with alcohol before re-staining. The bottom photograph is a close-up and higher magnification of the indicated section within the rectangle. The morphology of the specimen is now discernible and details of the morphology are necessary for proper evaluation / diagnosis of the biopsy.

[0056] [Figure 2c] The top photograph shows a microscopic image of the first half of an FNA biopsy (split into two halves) mounted on a chitosan-based material and stained with acridine orange without the addition of alcohol. The bottom photograph is a magnified and higher powered version of the indicated section within the rectangle. The morphology of the specimen is barely discernible.

[0057] [Figure 2d]The top photograph shows a microscopic image of the second half of the same FNA biopsy used for preparation of the sample shown in Figure 2c, which was mounted on a chitosan-based material, treated with alcohol, and then stained with acridine orange. The bottom photograph is a close-up and higher magnification of the indicated section within the rectangle. The morphology of the sample is now discernible, and details of the morphology are necessary for proper evaluation / diagnosis of the biopsy.

[0058] [Diagram 3] FIG. 3 is a plan view of a magnetic specimen holder comprising a base plate 302-1 with a mating magnetic element 304 and a support plate 302-2 with a magnetic element 303. The microscope sample is shown as a rectangular shaped chitosan-based material 300 onto which a biopsy sample is loaded. The chitosan-based material may have a recess to receive the biopsy sample.

[0059] [Figure 4] 4 is a schematic side view of a magnetic sample holder consisting of a base plate 402-1 and a support plate 402-2. A magnetic element 403 and a counter magnetic element 404 protrude from the base plate 402-1 and the support plate 402-2, respectively. A spacer 406 made of an elastic material is between the magnetic elements and the counter magnetic elements on either side of the plate, thus fixing the chitosan-based material 400 on which the biopsy sample 401 is loaded. The spacer 306 modulates the magnetic force of the magnetic elements 403 and 404 to protect the chitosan biopsy samples 400, 401 against compression. The chitosan material 400 and the biopsy sample 401 are placed with the top surface facing upwards on the support plate 402-2. The base plate 402-1 is coated with white paint and comprises a grip or handle section 405 which can be used to write notes on it or as a handle for the sample holder.

[0060] 4 further shows that the chitosan biopsies 400, 401 are mounted with a sample holder on a microscope stage 408 similar to the microscope stage of a VivaScope™ 2500 type microscope. A microscope objective 407 images the chitosan biopsies 400, 401 through a base plate 402-1. The plate holds the chitosan biopsies stationary during imaging relative to the objective and provides a flat surface for optical image sectioning.

[0061] [Figure 5a] FIG. 5 is a schematic diagram of a lattice box 501 with four compartments, each capable of receiving one slab of material. The lattice box is a commercially available biopsy processing or embedding cassette.

[0062] [Figure 5b] FIG. 5 shows a lattice box 501 housing a chitosan-based porous material in the form of a slab 500.

[0063] Unless otherwise stated, the following equipment, materials and chemicals are used:

[0064] The chitosan-based porous material used is CytoMatrix™ manufactured by UCS Diagnostic Srl. The CytoMatrix™ is used as a cell block with a size of about 1.2 cm x 1.2 cm (length x width) and a height of about 0.2 cm. For sample preparation, the CytoMatrix™ is placed in a biopsy processing or embedding cassette as shown in Figure 5b. Such cassettes are known and can be purchased under the trade names Swingsette™, Slimsette™ or Unisette™.

[0065] PBS Buffer: Phosphate buffered saline is prepared as follows: 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4 are added to 800 mL of distilled water. After mixing, the pH is adjusted to pH 7.4 using HCl. The mixture is further diluted with distilled water to obtain a final volume of 1 liter.

[0066] For staining, acridine orange is used (CAS No. 494-38-2).

[0067] A VivaScope™ 2500 confocal microscope from MAVIG GmbH is used for the examination.

[0068] A: Examination of body fluids such as exudates, urine, sputum and other cell suspensions Biopsies consisting of cell suspension samples are centrifuged at forces ranging from 50 RCF (relative centrifugal force, also known as "g") to 1500 RCF for 5 to 10 minutes. The time and centrifugal force depend on various parameters, such as cell type density and solution viscosity, and are adapted to the biopsy sample following known procedures.

[0069] The resulting cell pellet is collected and the supernatant is discarded. The cell pellet is resuspended and fixed for 30 seconds in a fixative with a 1:1 ratio of pellet volume to fixative volume. The fixative contains a 1:1 ratio of absolute ethanol to buffered formalin. Fixation is required to keep cell shape intact.

[0070] The suspension is centrifuged at 50-1500 RCF for 10 minutes. The pellet is collected and the supernatant is discarded. 50-100 μL of material from the cell pellet is loaded onto the CytoMatrix™, 2-3 drops (approximately 150 μL) of 70% ethanol in PBS buffer is added to the loaded material, and the mixture is incubated for approximately 10 seconds. Then, 2-3 drops (approximately 150 μL) of acridine orange are added onto the same spot. After an additional 20 seconds of incubation time to stain the sample, the microscope sample is rinsed with PBS buffer to remove excess dye.

[0071] The sample is mounted in a specimen holder and examined using a microscope capable of producing optical image sections, the evaluation of which is performed by software or by a human.

[0072] B: Use of fresh biopsy samples - sample preparation After loading a fresh biopsy sample onto the CytoMatrix™, 2-3 drops (approximately 150 μl) of 70% ethanol in PBS buffer are added onto the loaded material and the mixture is incubated for approximately 10 seconds. Then, 2-3 drops (approximately 150 μl) of acridine orange are added onto the same spot. After an additional 20 seconds of incubation time to stain the sample, the microscope sample is rinsed with PBS buffer to remove excess dye.

[0073] The loading of fine needle aspiration biopsies onto CytoMatrix™ is shown diagrammatically in FIG. 1a and a planar photograph of the sample for microscopic evaluation after alcohol treatment and staining is shown in FIG. 1b.

[0074] Fine needle biopsy (FNB) loading is shown diagrammatically in FIG. 1c, and a planar photograph of the sample for microscopic evaluation after alcohol treatment and staining is shown in FIG. 1d.

[0075] The sample is mounted in a specimen holder and examined using a microscope capable of generating optical image sections, the evaluation of which is performed by software or by a human.

[0076] C: Microscopy using confocal microscopy The sample is mounted on the specimen holder as shown in FIG. 4 by clamping the sample between two glass slides (402-1 and 402-2) that contain magnetic fastening means (403 and 404). The slides are magnetically attracted to each other, thus holding the sample (400 and 401) securely fixed between the slides. To avoid damage to the sample, a ductile material, here commercial Play-Doh manufactured by Hasbro, is placed between the magnets to increase the space between the slides and relieve the pressure on the sample resulting from the magnetic attraction.

[0077] The samples are imaged within minutes using a VivaScope™ 2500 microscope, which provides digitized images as optical sections that are evaluated by software or by humans.

[0078] D: Comparative example D.1: Two measurements are performed on the same fine needle aspiration biopsy sample, with and without the addition of alcohol.

[0079] Sample 1 (comparison) is prepared by loading the biopsy onto CytoMatrix™ according to the protocol given under section B, but without the addition of 70% ethanol in PBS buffer, followed by staining with acridine orange and rinsing with PBS buffer.

[0080] Following the protocol given under item B, sample 2 is prepared by using sample 1 after measurement by adding 70% ethanol in PBS buffer, followed by staining with acridine orange and rinsing with buffer.

[0081] Rinsing with PBS buffer removes excess stain from the samples.

[0082] Both samples were observed microscopically using a VivaScope™ 2500 confocal microscope from MAVIG GmbH, and the results are shown in Figure 2a (sample 1) and Figure 2b (sample 2). It can be clearly seen that the microscopic morphology is more pronounced in sample 2, which is prepared according to the present invention. Details of the microscopic morphology are necessary for proper evaluation / diagnosis.

[0083] D.2: Two measurements are performed on fine needle aspiration biopsy samples divided into two parts, either with or without the addition of alcohol.

[0084] Sample 3 (comparative) is prepared by loading one portion of the fine needle aspirate biopsy onto CytoMatrix™ following the protocol given under section B, but without the addition of 70% ethanol in PBS buffer, followed by staining with acridine orange and rinsing with PBS buffer.

[0085] Sample 4 is prepared by using the other part of the fine needle biopsy sample used in the sample preparation of Sample 3 by adding 70% ethanol in PBS buffer, followed by staining with acridine orange and rinsing with buffer according to the protocol given under section B.

[0086] Both samples were observed microscopically using a VivaScope™ 2500 confocal microscope from MAVIG GmbH, and the results are shown in Figure 2c (sample 3) and Figure 2d (sample 4). It can be clearly seen that the microscopic morphology is more pronounced in sample 4, which is prepared according to the present invention. [Explanation of symbols]

[0087] 100 = Chitosan-based materials 101=Fine Needle Aspiration (FNA) Biopsy Sample 102=Syringe for fine needle aspiration biopsy 103 = Biopsy specimen with tubular structures such as those obtained using fine needle biopsy (FNB) 104 = Syringe for core biopsy, preferably for fine needle biopsy 201 = Fine needle aspiration (FNA) biopsy sample loaded onto CytoMatrix™ 300 = Chitosan-based materials 301=Biopsy sample 302-1 = base plate with mating magnetic element 304 302-2 = Support plate with magnetic element 303 303=Magnetic element 304=Mating magnetic element 305=Grip or handle section 400 = Chitosan-based materials 401=Biopsy sample 402-1=base plate 402-2=support plate 403=Magnetic element 404=Mating magnetic element 405=Grip or handle section 406=Spacer 407=Objective lens from a microscope 408=Sample holder on microscope stage 500 = Chitosan-based porous material in the form of a slab 501=Grid Box

Claims

1. 1. A method for preparing a sample for microscopy, comprising the steps of: Step (i): Using a solid or ductile chitosan-based porous material having interconnected pores with a size between 5 μm and 700 μm and a total porosity (volume fraction) between 40% and 90%; Step (ii): Adding at least one biopsy sample containing tissue and / or eukaryotic and / or prokaryotic cells and / or eukaryotic cells containing viral particles to said chitosan-based porous material; and Step (iii): (C １ -C ６ )-Adding an alkyl alcohol or a mixture thereof A method comprising:

2. 2. The method according to claim 1, wherein the chitosan-based porous material is obtained by preparing a solution of chitosan and / or lactosylated chitosan or a solution of vinyl derivatives of chitosan, the vinyl derivatives being alone or mixed with sulfhydryl derivatives of chitosan, all compounds having a molecular weight between 50 kDa and 200 kDa, dissolved in an acid solution of a polar inorganic or organic acid, followed by gelation of the solution to obtain a hydrogel, and freeze-drying the resulting hydrogel to obtain the chitosan-based porous material.

3. (C １ -C ６ 2. The process of claim 1, wherein the .alpha.-alkyl alcohol or mixture thereof used in step (iii) contains at least 5% water or an aqueous buffer solution.

4. 10. The method of claim 1, further comprising step (iv), staining the biopsy sample by adding at least one fluorescent dye.

5. 5. The method of claim 4, using 2,3-benzoquinoline type dyes, acridine orange, acriflavine, proflavine and acridine yellow, thiazine type dyes, toluidine blue and methylene blue, Masson's trichrome staining colour, methyl blue, aniline blue, fast green FCF, water blue, hematoxylin, eosin, ethidium bromide, rhodamine 123, cyber green, acid fusion, sirius red, Col-F, Nile blue, Nile red, oil red O, auramine O, neutral red, patent blue, fluorescein, indocyanine green (ICG), methyl green, pyronin Y and combinations thereof, the dyes may be attached directly or via a linker such as an antibody or a lectin.

6. 2. The method of claim 1, wherein the chitosan based porous material is used in the form of a slab or block, and at least one surface of the slab or block is capable of receiving the biopsy sample in its entirety when collected through incisional biopsy, stereotactic biopsy, sternal biopsy, chorionic villus biopsy, cone biopsy, endoscopic biopsy, needle biopsy (percutaneous biopsy), fine needle aspiration biopsy (FNA), fine needle biopsy (FNB), small needle biopsy, microneedle biopsy, core needle biopsy or cell suspension biopsy.

7. 7. The method of claim 6, wherein the length and / or width or diameter of the at least one surface capable of receiving the biopsy sample is 1.5 cm or less.

8. 7. The method of claim 6, further comprising step (v) mounting the biopsy sample on a specimen holder to be used in microscopy.

9. The method of claim 8, wherein the sample is sandwiched between a first plate 302-1 and a second plate 302-2 that are provided with a window through which an optical beam passes.

10. 10. The method of claim 9, wherein the first plate 302-1 and the second plate 302-2 are provided with magnetic elements 303 and 304 to ensure that the sample is fixed between the plates and without damaging the biopsy sample.

11. 2. The method of claim 1, wherein a fresh histopathological biopsy sample is used in step (ii).

12. 12. The method of claim 11, wherein the histopathological biopsy sample is collected using at least one of the following biopsy methods: incisional biopsy, slice biopsy, brush biopsy, swab biopsy, stereotactic biopsy, sternal biopsy, chorionic villus biopsy, cone biopsy, endoscopic biopsy, adhesive patch-based biopsy, needle biopsy (percutaneous biopsy), fine needle aspiration biopsy (FNA), fine needle biopsy (FNB), small core needle biopsy, microcore needle biopsy, and core needle biopsy.

13. A sample prepared according to the method of any one of claims 1 to 12.

14. Use of a sample prepared by the method of any one of claims 1 to 12 for immediate examination using optical image sections of said biopsy sample after collection.

15. Use of a sample prepared by the method of any one of claims 1 to 12 for laser scanning confocal microscopy, multiphoton fluorescence microscopy, two-photon fluorescence, three-photon fluorescence, optical coherence tomography and full-field optical coherence tomography, microscopy with ultraviolet sectioning excitation, high and ultra-high resolution subsurface imaging, stimulated Raman scattering, stimulated Raman diffusion tissue diagnostics, multi-harmonic generation microscopy, second harmonic generation (SHG) or third harmonic generation (THG).

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