Apparatus and method for cytopathological staining - Patents.com

Abstract

Disclosed herein is an automated platform, device and method of use thereof for multiplex staining and preparation of cytological slides of biological specimens from patients, such as patients undergoing evaluation for cancer diagnosis. Multiplex staining of atypical cells in biological specimens can improve sensitivity and specificity for confidently determining the presence of pathological cells. The device and method disclosed herein provide an efficient, high-throughput approach for cytopathological examination of patient specimens.

Description

[Technical field]

[0001] Aspects of the present disclosure relate generally to an automated platform for cytopathological staining of atypical cells, including cancer. [Background technology]

[0002] Cytopathology is a science that examines the causes and pathogenesis of diseases, as well as changes in the physiological functions of cells at the onset of diseases that are mainly based on abnormal conditions within the cells, and provides basic information for the diagnosis, prevention, and treatment of diseases. Clinical applications include exfoliative cytology, fine needle aspiration cytology, circulating tumor cytology, and other cytological diagnoses (e.g., surgical cytology, bone marrow and peripheral blood cytology, AIDS cytology, etc.).

[0003] The positive determination of atypical cells, such as cancer cells, may vary depending on the interpretation of an experienced pathologist. To ensure reliable interpretation and diagnosis, biological specimens containing cells suspected to be atypical must be properly processed, which usually involves staining the cells to improve the detection of morphological and / or functional characteristics. Furthermore, pathologists can benefit from more efficient operations without compromising the reliability of cytopathological specimen preparation. Therefore, there is a need for improved equipment or devices for specific rapid cytopathological diagnosis, and methods for using the same. Summary of the Invention [Means for solving the problem]

[0004] Disclosed herein is an apparatus for performing multiplex cytopathological staining. An embodiment of the apparatus of the present invention includes at least a staining vessel configured to receive aqueous cell culture medium, staining reagents, washing reagents, and / or slide mounting medium, and cells from a biological specimen obtained from a patient, and an extraction vessel configured to receive spent aqueous cell culture medium, spent staining reagents, and / or spent washing reagents. The staining vessel and the extraction vessel are operatively separated via a semipermeable membrane that is permeable to the aqueous cell culture medium, staining reagents, washing reagents, and / or mounting medium, but not to the cells, thereby retaining the cells in the staining vessel. Operation of the apparatus disclosed herein allows for multiple staining and washing steps to be performed without significant cell loss, and the staining and washing steps may be performed in a variety of ways, including centrifugation and resuspension. In this manner, the cell specimen can be stained in multiple ways, thereby improving the positive determination of atypical cells in the biological specimen for cytological diagnosis, such as diagnosing a disease.

[0005] Also disclosed herein are methods of using the devices of the present invention to perform multiplex cytopathological staining.

[0006] Also disclosed herein is a pathology slide assembly. An embodiment of the pathology slide assembly of the present invention includes at least a pathology slide and a pathology coverslip configured to be stacked together and sealed to define an interior void accessible through an inlet port and an outlet port of the pathology slide assembly. The pathology slide may be coated with a cell adhesive material that aids in the attachment and retention of cells from a biological specimen within the interior void. A surface of the pathology coverslip may define an interior void within the pathology slide assembly, which may be a single "pond" type reservoir or may be configured to include one or more microchannels.

[0007] Also disclosed herein is a multiple pathology slide assembly comprising two or more pathology slides as disclosed herein joined together, which may improve capture of cells in a specimen by providing a larger surface area for specimen flow and cell encapsulation.

[0008] Also disclosed herein are methods of making the pathology slide assemblies disclosed herein and methods of using the pathology slide assemblies disclosed herein. The cells enclosed in the pathology slide assemblies provided herein may be stained or otherwise processed by the apparatus for multiplex cytopathological staining disclosed herein.

[0009] Further disclosed herein is an apparatus for automating the cell staining process.

[0010] Embodiments of the present disclosure provided herein are illustrated by the following numbered embodiments.

[0011] 1. An apparatus for performing multiple cytopathological staining, comprising: a) a staining vessel configured to receive an aqueous cell culture medium, a staining reagent, a washing reagent and / or a slide mounting medium, and cells from a biological specimen obtained from a patient, and configured to provide stained cells by alternating staining and washing steps; b) an extraction vessel configured to receive and remove the used aqueous cell culture medium, the used staining reagent and / or the used washing reagent from the staining vessel; Including, the staining vessel and the extraction vessel are operably separated via a semipermeable membrane, the semipermeable membrane being permeable to the aqueous medium for the cells, the staining reagent, the washing reagent and / or the mounting medium, but not to the cells; Device.

[0012] 2. The device of embodiment 1, wherein the semi-permeable membrane comprises pores configured to allow liquids and small molecules to pass through the semi-permeable membrane but not allow the cells to pass through.

[0013] 3. The device of embodiment 2, wherein the diameter of each pore is 0.05 μm, 0.1 μm, 0.2 μm, 0.22 μm, 0.3 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 3 μm, 5 μm or 10 μm, or a pore diameter within a range defined by any two of these values.

[0014] 4. The device of any one of embodiments 1 to 3, wherein the staining reagent comprises a cytomorphological stain, an immunocytochemical stain, or a chromogenic in situ hybridization stain, or any combination thereof.

[0015] 5. The device of embodiment 4, wherein the cytomorphological stain comprises Diff-Quick stain, Papanicolaou stain, Wright-Giemsa stain, Hematoxylin / Eosin stain, or any derivative or modification thereof.

[0016] 6. The device according to any one of embodiments 1 to 5, wherein the semipermeable membrane is configured to move the staining reagent used in each staining step in the staining container and the washing reagent used in each washing step to the extraction container by passive diffusion, or is configured to move the staining reagent and the washing reagent to the extraction container when the extraction container is under negative pressure relative to the staining container, and the negative pressure may be applied by an extraction pump.

[0017] 7. The device according to any one of embodiments 1 to 6, wherein the staining vessel and the extraction vessel are configured to be separated or blocked by a shutter that prevents or inhibits the aqueous cell medium, the staining reagent and / or the washing reagent from moving through the semipermeable membrane between the staining vessel and the extraction vessel.

[0018] 8. The apparatus of any one of embodiments 1 to 7, further comprising a reagent source configured to provide the aqueous cell medium, the staining reagent, the washing reagent and / or the slide mounting medium to the staining vessel, the reagent source may be operably connected to a reagent pump configured to move the aqueous cell medium, the staining reagent, the washing reagent and / or the slide mounting medium from the reagent source to the staining vessel, and the aqueous cell medium may contain the cells.

[0019] 9. An apparatus as described in any one of embodiments 1 to 8, further comprising a specimen collection flow path configured to collect the stained cells, e.g., for purposes of mounting, wherein the stained cells may be contained in the slide mounting medium, and wherein the specimen collection flow path is configured to collect the stained cells using negative pressure.

[0020] 10. A method for multiplex cytopathological staining of cells derived from a biological specimen obtained from a patient, comprising staining the cells by one or more staining steps using an apparatus described in any one of embodiments 1 to 9.

[0021] 11. The method of embodiment 10, wherein the staining step comprises staining the cells with a cytomorphological stain, an immunocytochemical stain, or a chromogenic in situ hybridization stain, or any combination thereof.

[0022] 12. The method according to embodiment 10 or 11, wherein the cytomorphological stain comprises Diff-Quick stain, Papanicolaou stain, Wright-Giemsa stain, Hematoxylin / Eosin stain, or any derivative or modification thereof.

[0023] 13. A pathology slide assembly comprising a pathology slide and a pathology coverslip, the pathology slide and the pathology coverslip are configured to be sealed together; the pathology slide and the pathology coverslip defining an interior cavity accessible through an inlet port and an outlet port of the pathology slide assembly; the internal cavity is configured to receive a suspension of cells from a biological specimen obtained from a patient through an inlet port and to discharge medium free of the cells through an outlet port. Pathology slide assembly.

[0024] 14. A pathology slide assembly as described in embodiment 13, wherein the thickness of the pathology slide assembly is compatible with an optical microscope.

[0025] 15. A pathology slide assembly as described in embodiment 13 or 14, wherein the surface of the pathology slide defining the internal cavity comprises a cell adhesive material, which may be an adhesive gel, a protein coating, a sugar coating or a nanomaterial configured to immobilize the cells.

[0026] 16. A pathology slide assembly according to any one of embodiments 13 to 15, wherein the internal cavity comprises a single reservoir of uniform shape.

[0027] 17. A pathology slide assembly described in any one of embodiments 13 to 15, wherein the pathology coverslip is configured to include one or more microchannels, the internal cavity includes a concave space defined by the one or more microchannels, and the one or more microchannels are configured to allow the cells to pass therethrough.

[0028] 18. A pathology slide assembly according to any one of embodiments 13 to 17, wherein a surface of the pathology slide or a surface of the coverslip, or both, preferably the surface of the pathology slide defining the internal void, is nano-roughened, and the nano-roughened surface enhances capture of the cells in the suspension.

[0029] 19. A pathology slide assembly described in any one of embodiments 13 to 18, wherein the inlet port is configured to be operably connected to an outlet port of a second pathology slide assembly via a connecting flow path, and / or the outlet port is configured to be operably connected to an inlet port of a third pathology slide assembly via another connecting flow path.

[0030] 20. A multiple pathology slide assembly comprising two or more pathology slide assemblies according to any one of embodiments 13 to 19, the two or more pathology slide assemblies are connected in series via an inlet port and an outlet port of each pathology slide assembly, such that internal cavities of the two or more pathology slide assemblies are in fluid communication with each other to form a continuous space; an exposed inlet port of one of the two or more pathology slide assemblies and an exposed outlet port of another of the two or more pathology slide assemblies are not connected to another port, allowing access to the continuous space; Multiple pathology slide assembly.

[0031] 21. A method for encapsulating cells derived from a biological specimen obtained from a subject using a pathology slide assembly according to any one of embodiments 13 to 19, or a plurality of pathology slide assemblies according to embodiment 20, comprising: a) flowing the suspension of cells into an interior cavity of the pathology slide assembly through an inlet port of the pathology slide assembly; or b) allowing the cell suspension to flow through an exposed inlet port of the plurality of pathology slides into the continuous space of the plurality of pathology slides; precipitating and / or adhering said cells to a surface of an internal cavity defined by said pathology slide or said plurality of pathology slides; A method comprising:

[0032] 22. The method further comprises the step of flowing a washing reagent and / or a mounting medium through an inlet port of the pathology slide assembly into the internal cavity of the pathology slide assembly (or through exposed inlet ports of the plurality of pathology slide assemblies into the continuous space of the plurality of pathology slide assemblies) to exchange liquid components in the suspension of cells; the cells are retained within the interior void; the liquid components in the suspension are expelled through an exit port (or an exposed exit port of the plurality of pathology slides assembly); 22. The method of embodiment 21.

[0033] 23. The method of embodiment 21 or 22, further comprising imaging the cells precipitated and / or attached to the surface of the pathology slide or the plurality of pathology slides by optical microscopy.

[0034] 24. The method according to any one of embodiments 21 to 23, wherein the cells are fixed and / or permeabilized.

[0035] 25. The method according to any one of embodiments 21 to 24, wherein the cells are stained by cytomorphological staining, immunocytochemical staining, or chromogenic in situ hybridization staining, or any combination thereof, and the staining of the cells may be performed using an apparatus according to any one of embodiments 1 to 9, or a method according to any one of embodiments 10 to 12.

[0036] 26. A combined dyeing apparatus comprising: a) a pathology slide assembly according to any one of claims 13-19 and an apparatus according to any one of claims 1-9, wherein the specimen collection channel of the apparatus is configured to be operably connected to an inlet port of the pathology slide assembly; or b) a plurality of pathology slide assemblies according to embodiment 20; and an apparatus according to any one of embodiments 1 to 9, wherein a specimen collection channel of the apparatus is configured to be operably connected to an exposed inlet port of the plurality of pathology slide assemblies. Combination dyeing equipment.

[0037] 27. The device described in any one of embodiments 1 to 9, the method described in any one of embodiments 10 to 12, the pathology slide assembly described in any one of embodiments 13 to 18, the method described in any one of embodiments 19 to 23, or the combination staining device described in embodiment 26, wherein the cells derived from the biological specimen obtained from the patient include atypical cells, and the atypical cells may be cancer cells.

[0038] 28. The apparatus, method, pathology slide assembly, method or combined staining apparatus of embodiment 24, wherein the cells comprise urine sediment cells.

[0039] 29. The device, method, pathology slide assembly, method or combined staining device of embodiment 25, wherein one or more bladder cancer specific biomarkers are stained in the urine sediment cells, and the one or more bladder cancer specific biomarkers may be selected from the group consisting of S100P, p63, M344, LDQ10, 19A211, GATA-3, Ki-67, p16, Her-2, PD-L1, CTLA4, CK-17, CK-20, nmp-22, bladder tumor antigen (BTA), hTERT and minichromosome maintenance protein 5 (MCM5).

[0040] 30. A combination staining apparatus, comprising a sample inlet, an appearance inspection system, an operation status indicator light, a control panel, a slide glass inlet, a mouse and a keyboard.

[0041] 31. The apparatus of embodiment 30, further comprising a reagent dispensing module, a staining chamber with a porous membrane, a shaker, a visual inspection field, a device for removing the chamber walls, a device for transferring stained cells to a slide, a waste collection hole, and a liquid waste collection mechanism.

[0042] 32. The apparatus of embodiment 30, wherein the liquid waste collection system further comprises an aspirator.

[0043] 33. The method of embodiment 30, wherein the process of preparing stained pathology slides from liquid specimens is automated, e.g., automated in a closed system.

[0044] 34. The method of embodiment 31, wherein the processing of the sample is performed for about 3 hours.

[0045] 35. A dyeing chamber or dyeing vial having a removable cylindrical wall, a) an upper portion; b) a porous membrane; c) Lower and Including, the lower portion is connected to an air pump and a used liquid reagent reservoir, and the upper portion is configured for removably coupling to the lower portion; the porous membrane is disposed between the upper and lower portions; the upper portion includes an overhanging lip portion that extends beyond the juncture of the porous membrane and the lower portion, the lip portion contacting a shelf portion of the lower portion; Staining chamber or staining vial.

[0046] 36. A dyeing chamber or dyeing vial according to embodiment 35, wherein the porous membrane is circular or elliptical, with a maximum size of 20 mm x 20 mm if circular and a maximum size of 20 mm x 40 mm if elliptical.

[0047] 37. The staining chamber or staining vial of embodiment 35, further comprising a seal around the periphery of the lower portion above the shelf, the seal configured to contact a surface of the upper portion.

[0048] 38. The staining chamber or staining vial of embodiment 37, wherein the seal comprises plastic or rubber.

[0049] 39. The staining chamber or vial of embodiment 35, further comprising a protruding rim around the top of the upper portion.

[0050] 40. A dyeing chamber or dyeing vial according to embodiment 39, wherein the protruding rim serves as a fastener for pulling out the cylindrical wall of the dyeing chamber, which is discarded after completion of the dyeing process.

[0051] 41. A staining chamber or staining vial as described in embodiment 40, wherein the cylindrical wall of the staining chamber is removed to expose the stained cells, which are then pressed onto a slide containing a cell adhesive material.

[0052] 42. A staining chamber or staining vial according to embodiment 41, wherein the step of migrating the cells is facilitated by passing positive air pressure underneath the porous membrane.

[0053] 43. A staining chamber or staining vial according to any one of embodiments 35 to 42, wherein the staining chamber is configured so that used reagents can be sucked downwardly into the lower portion and discarded, and the upper portion is configured so that new reagents can be added.

[0054] 44. A staining chamber or staining vial according to any one of embodiments 35 to 43, configured such that stained cells are exposed after the top is removed.

[0055] 45. A method for detecting bladder cancer from a biological specimen obtained from a subject using the device according to any one of embodiments 30 to 32, comprising: i. introducing at least one cell-containing liquid specimen into said device; ii. staining said at least one cell-containing liquid specimen; iii. transferring the stained cells to a pathology slide; iv. fixing the stained cells; and v. imaging the stained specimen slides to detect analytes indicative of cancer biomarker expression and / or cytopathology. A method comprising:

[0056] 46. ​​The method of embodiment 45, wherein the staining step comprises a combination of cytomorphological staining and staining for a cancer biomarker.

[0057] 47. The method of embodiment 45, wherein the staining, transferring and fixing steps are performed by the device.

[0058] 48. The method of embodiment 45, wherein the cells in the liquid specimen comprise urine sediment cells.

[0059] 49. The method of embodiment 45, wherein the staining step comprises cytomorphological staining, including Diff-Quick staining, Papanicolaou staining, Wright-Giemsa staining, hematoxylin / eosin staining, or derivatives or modifications thereof.

[0060] 50. The method of embodiment 45, wherein the staining step comprises staining a cancer biomarker, the cancer biomarker comprising a bladder cancer biomarker, and the bladder cancer specific biomarker is selected from the group consisting of S100P, p63, M344, LDQ10, 19A211, GATA-3, Ki-67, p16, Her-2, PD-L1, CTLA4, CK-17, CK-20, nmp-22, bladder tumor antigen (BTA), hTERT and minichromosome maintenance protein 5 (MCM5).

[0061] 51. The method of embodiment 45, wherein the evaluation of the stained specimen is performed using an optical or fluorescent microscope.

[0062] 52. The method of embodiment 45, wherein the cells are stained by cytomorphological staining, immunocytochemical staining, or chromogenic in situ hybridization staining, or any combination thereof.

[0063] 53. A method for detecting bladder cancer from a biological specimen obtained from a subject, wherein the device described in any one of embodiments 30 to 32 further comprises a staining chamber or staining vial described in any one of embodiments 35 to 44.

[0064] 54. The device according to embodiment 31, wherein the dyeing chamber provided with a porous membrane is a dyeing chamber according to embodiments 35 to 44.

[0065] 55. An apparatus for performing multiple cytopathological staining, comprising: one or more staining chambers including a porous membrane having a surface configured to retain cells; a visual inspection system configured to determine completion of the dyeing process; a reagent dispensing module configured to dispense reagent to the one or more staining chambers; a shaker disposed beneath the one or more dyeing chambers; and a transfer arm configured to transfer stained cells from the one or more staining chambers to one or more glass slides. 13. An apparatus comprising:

[0066] 56. The device of embodiment 55, wherein the one or more staining chambers include an upper portion configured to accommodate the reagent, the upper portion being configured to be separable from the porous membrane and a lower portion of the staining chamber.

[0067] 57. The apparatus of embodiment 56, comprising a gripper configured to engage the upper portion and remove the upper portion from the porous membrane and the lower portion.

[0068] 58. The apparatus of embodiment 57, wherein the gripper is configured to engage with the upper lip portion.

[0069] 59. The apparatus of embodiment 57 or 58, wherein the gripper is configured to move the upper portion to a waste collection hole.

[0070] 60. The apparatus of any one of embodiments 55-59, comprising a pump configured to generate pressure below the porous membrane.

[0071] 61. An apparatus according to any one of embodiments 55 to 60, comprising a rail configured to transport the shaker and the one or more staining chambers from a first position within the apparatus to a second position within the apparatus, and the transport arm configured to transfer the stained cells from the porous membrane to the one or more glass slides when the one or more staining chambers are positioned in the second position.

[0072] 62. An apparatus described in any one of embodiments 55 to 61, wherein the visual inspection system includes a camera in electronic communication with a hardware processor, the camera configured to image at least a portion of the staining chamber, and the hardware processor configured to determine the completion of the staining process based at least in part on the image generated by the camera.

[0073] 63. The apparatus of any one of embodiments 55-62, further comprising a liquid waste collection device, the liquid waste collection device being in fluid communication with a lower portion of the one or more dyeing chambers, the lower portion of the one or more dyeing chambers facing the surface of the porous membrane.

[0074] 64. The apparatus of embodiment 63, wherein the liquid waste collection device includes a pump configured to generate negative pressure within a lower portion of the one or more dyeing chambers. [Brief description of the drawings]

[0075] Further features and modifications other than those described above will be readily apparent from the following description of the drawings and the embodiments illustrated therein. The following drawings are illustrative of the embodiments and are not intended to limit the scope of the present invention.

[0076] [Figure 1A-C] 1 illustrates an exemplary cytopathology staining device embodiment in a "vertical" configuration.

[0077] [Fig. 1D-F] 1 illustrates an exemplary cytopathological staining device embodiment in a "horizontal" configuration.

[0078] [Figure 2A] FIG. 2C shows one embodiment of an exemplary pathology slide that can be used with the pathology coverslip shown in FIG. 2B or other pathology coverslip embodiments disclosed herein.

[0079] [Figure 2B] 2B shows an exemplary pathology coverslip embodiment that can be used with the pathology slide shown in FIG. 2A or other pathology slide embodiments disclosed herein.

[0080] [Figure 2C] FIG. 2A-B show an exemplary embodiment of a pathology slide assembly that may be comprised of an embodiment of a pathology slide and an embodiment of a pathology coverslip, or other pathology slide and pathology coverslip embodiments disclosed herein.

[0081] [Figure 2D]2C, or other pathology slide assemblies disclosed herein.

[0082] [Figure 3A-B] 3A-3C show embodiments in which two or more of the exemplary pathology slide assemblies disclosed herein are joined together. As shown in FIG. 3B, combinations of the same or different types of exemplary pathology slide assemblies may be joined together.

[0083] [Figure 4A] 1 illustrates an embodiment of an exemplary cytopathology staining apparatus in a "vertical" configuration operably coupled to an exemplary pathology slide assembly disclosed herein via a specimen collection channel.

[0084] [Figure 4B] 1 illustrates an embodiment of an exemplary cytopathology staining apparatus in a "horizontal" configuration operably coupled to an exemplary pathology slide assembly disclosed herein via a specimen collection channel.

[0085] [Figure 5A-B] 1 illustrates another exemplary staining chamber embodiment in an assembled form.

[0086] [Figure 6A-B] 1 illustrates a cross-section of another exemplary staining chamber embodiment in exploded form.

[0087] [Figure 7A-B] 1 illustrates a cross-section of another exemplary dye chamber embodiment in exploded form, showing details of the connection between the upper and lower portions.

[0088] [Figure 8] 1 illustrates a cross-section of another exemplary dye chamber embodiment in assembled form, showing details of the connection between the upper and lower portions.

[0089] [Figure 9] The external appearance of the dyeing device is shown.

[0090] [Figure 10] FIG. 1 shows an internal view of the staining apparatus showing the reagent dispensing module, staining chamber and shaker.

[0091] [Figure 11] An internal view of the staining apparatus showing the visual test field is shown.

[0092] [Figure 12] FIG. 1 shows an internal view of the staining apparatus showing the device for removing the chamber walls.

[0093] [Figure 13] FIG. 1 shows an internal view of the staining apparatus showing the device that transfers the stained cells to the slide.

[0094] [Figure 14] FIG. 1 shows an internal view of the staining apparatus showing the waste collection holes / reservoirs.

[0095] [Figure 15] FIG. 2 shows an internal view of the staining apparatus showing the liquid waste recovery mechanism. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0096] Disclosed herein are devices and methods of using the devices for multiplex staining of biological specimens and preparation of cytological slides, such as for cytopathological diagnosis of diseases involving cellular atypia, including but not limited to cancer. The approaches provided herein ensure consistent and uniform staining for analysis by staining cells from biological specimens in suspension. The devices provided herein may include a membrane that is permeable to reagents and stains but not to cells. The devices and methods of using the devices disclosed herein may include automation, which advantageously reduces variability in specimen preparation due to human error.

[0097] "Cytopathology" refers to the examination of individual cells from a patient's biological specimen to assess their morphological characteristics and possible atypicality, such as for the diagnosis of various diseases. Such assessment can be useful in the diagnosis of cancer, since cancer cells exhibit atypical features such as unregulated division; abnormal genomes and enlarged nuclei; changes in cell morphology, cell arrangement and internal pH; and significant changes in functional phenotypes such as expression of cell markers and other proteins.

[0098] Conventional cytopathological stains include the Papanicolaou stain (Pap stain), Wright-Giemsa stain, and Diff-Quick stain. The Diff-Quick stain is the most widely used stain in routine cytopathological practice and is recommended by the World Health Organization (WHO). It usually uses xanthene dyes (e.g., eosin Y) and thiazine dyes (e.g., methylene blue or azure A). These dyes can clearly show nuclear features, such as the shape and size of the nucleus and nucleoli; chromatin density; cytoplasmic components, such as mucin, lipid droplets, and neurosecretory granules; and cell membrane features, such as membrane grooves, processes, and vacuoles. They can also stain extracellular materials, such as free mucin, colloids, and ground substance. Such features may be useful for cytopathological diagnosis.

[0099] Cells may be fixed and mounted on a slide before staining. However, this method can have staining "blind spots" where the surface of cells mounted on the slide may not be properly stained, and / or the staining dye may be trapped between the surface of one cell and the surface of another cell, despite washing steps, resulting in the so-called "edge effect." Both of these issues can affect the accuracy of cytopathology detection.

[0100] Moreover, such conventional methods may provide unreliable information. When target cells are difficult to isolate or the number of target cells in a biological specimen is low (such as in the case of urinary sediment cells for bladder cancer diagnosis), the low number of cells available for testing may affect the reliability of the diagnosis of atypical cells. To improve the success rate (e.g., to reduce false negatives and false positives), the detection of biomarkers may be combined with conventional cell morphological staining using methods such as immunocytochemical staining or chromogenic in situ hybridization. Identification of biomarkers for cells that are also characterized as atypical cells can significantly improve the positive diagnosis by pathologists, even in patient specimens with low cell numbers. However, care must be taken during the sequential processing of biological specimens for morphological examination and staining of biomarkers to limit cell loss and avoid damage to the cell morphology at the start of the test at each step.

[0101] Multiplex staining of urinary sediment cells for bladder cancer detection Urine cytology is the evaluation of urine sediment cells (and malignant cellular components) on a slide by a pathologist, who examines the cytopathological characteristics of individual cells by conventional light microscopy. Urine cytology is routinely used in clinical practice as a noninvasive test for bladder cancer. Bladder cancer can be classified as low-grade or high-grade. Low-grade tumors are characterized by slow proliferation of cancer cells and cytomorphological similarity to normal urothelial cells and other bladder cells. Although low-grade bladder cancers are less likely to progress to more aggressive or invasive tumors than high-grade bladder cancers, early detection and preservation of low-grade tumors may be desirable, as their presence may indicate a predisposition to developing aggressive bladder cancer. High-grade tumors may contain cells that grow rapidly and invade the surrounding bladder muscle, which may eventually result in metastasis. Although there are accepted pathological methods for detecting high-grade bladder tumors, low-grade tumors can be difficult to detect due to their similarity to normal bladder cells. Thus, although urine cytology is highly effective in detecting high-grade and high-stage bladder disease with high sensitivity and specificity, it has been found to be less effective as a means of detecting low-grade tumors, with a reported sensitivity of only 4-31%.

[0102] Traditionally, a 20-25 mL urine sample is centrifuged to collect urinary sediment cells, which are then placed in a thin layer on a pathology slide and stained with clinically accepted methods to reveal cellular morphological characteristics that aid the pathologist in their diagnosis.

[0103] Bladder cancer grade refers to the degree to which cancer cells resemble healthy cells when stained and viewed under a microscope. However, low-grade bladder cancer cells can be misclassified as normal cells or classified as atypical or suspicious cells without a definitive diagnosis due to their relatively normal appearance. Urinary cytology is clearly inadequate for detecting low-grade bladder tumors and has a very high false-negative rate, so other technologies to supplement urinary cytology are being explored. Therefore, the expression of bladder tumor-specific biomarkers in exfoliated malignant tumor cells has attracted attention. To meet this growing need, two major technology platforms have been developed: 1) Quantification of bladder tumor-specific biomarker protein concentrations in urine samples 2) Detection of biomarker expression in urinary sediment cells by fluorescent methods, i.e., immunofluorescence staining (IF) and fluorescence in situ hybridization (FISH)

[0104] The U.S. Food and Drug Administration (FDA) has approved six products for the detection of bladder tumors in urine specimens. These products are BTA stat, BTATRAK, NMP22 BC, NMP22 BladderChek, ImmunoCyt / uCyt, and UroVysion. However, these tests are not considered superior to urine cytology due to suboptimal sensitivity and specificity, and are not widely adopted by urologists. The first four of the aforementioned tests (BTA stat, BTATRAK, NMP22 BC, and NMP22 BladderChek) measure the protein concentration of bladder cancer-specific biomarkers, while ImmunoCyt / uCyt and UroVysion detect the biomarkers by immunofluorescence. None of these tests provide detailed cytomorphological information that is suitable for pathology diagnosis. Although immunofluorescence staining can show the overall shape and size of the target cells, it is insufficient and unsuitable for pathologists to make a morphological diagnosis. Therefore, these methods do not provide detailed morphological or cytopathological information sufficient for a pathologic diagnosis.

[0105] Furthermore, FDA-approved methods have large interobserver variability in sensitivity and specificity that can affect the accuracy of the tests, including a high number of false-positive results due to benign or other causes, such as infection, stones, hematuria, and recent instrumentation of the urinary system. Additionally, the need for specialized laboratory equipment, such as darkrooms and expensive fluorescence microscopes, as well as skilled readers to interpret the test results, limits the widespread use of these assays.

[0106] Combining cytomorphological assessment with cancer biomarker assessment overcomes these limitations and allows for more reliable identification of low-grade and high-grade bladder cancer in patient samples. In this method, cells are stained in suspension rather than after attachment to a slide, suppressing undesirable staining artifacts. This combined cytomorphological assessment with cancer biomarker assessment method is described in Chinese patent applications CN202010468262.6 and CN202010467313.3, both of which are expressly incorporated herein by reference in their entirety. The methods described in these publications are compatible with the apparatus and methods disclosed herein and can be operated and performed manually, as well as automated to further improve the consistency of specimen preparation.

[0107] Exemplary diagnostic cytomorphological stains that may be used with the devices and methods disclosed herein for the diagnosis of bladder cancer include, but are not limited to, Diff-Quick stain, Papanicolaou stain, Wright-Giemsa stain, hematoxylin / eosin stain, or derivatives or modifications thereof. Exemplary bladder cancer-specific biomarkers that may be used for immunocytochemical or chromogenic in situ hybridization staining in the devices and methods disclosed herein include, but are not limited to, S100P, p63, M344, LDQ10, 19A211, GATA-3, Ki-67, p16, Her-2, PD-L1, CTLA4, CK-17, CK-20, nmp-22, BTA, hTERT, or MCM5.

[0108] definition In the following detailed description, the present invention will be described with reference to the accompanying drawings, which form a part hereof. Unless otherwise specified, similar symbols in the drawings generally indicate similar components. The embodiments described in the detailed description, drawings, and claims are intended to be illustrative and are not intended to limit the present invention in any way. Furthermore, other embodiments are possible and other changes are possible without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure as outlined herein and illustrated in the drawings can be arranged, substituted, combined, separated, and designed in various configurations, and all such aspects are expressly encompassed herein.

[0109] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs when interpreted in light of this disclosure. For purposes of the present disclosure, the following terms are defined below.

[0110] The disclosure generally uses positive language to describe various embodiments of the invention, and includes embodiments in which all or part of the subject matter of the invention, such as substances or materials, method steps and conditions, protocols or procedures, is excluded.

[0111] As used herein, the articles "a" and "an" are used to refer to one or to more than one (e.g., to at least one) of the grammatical object of the article. For example, "an element" means one component or more than one component.

[0112] "About" means that a particular quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length varies by about 10% from the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0113] Throughout this specification, unless otherwise stated, the terms "comprise" and "comprising" are meant to include the steps or components or steps or components described herein, but not to exclude other steps or components or steps or components. "Consisting of" means to include only those listed before this term. Thus, the term "consisting of" means that the components listed before this term are necessary or essential, and other components may not be included. "Consisting essentially of" means to include the components listed before this term, and also includes other components that do not interfere with or contribute to the activity or action described in connection with the disclosure of these components. Thus, the term "consisting essentially of" means that the components listed before this term are necessary or essential, but other components are optional and may or may not be included depending on whether they have a substantial effect on the activity or action of the components listed before this term.

[0114] As used herein, the terms "individual", "subject" or "patient" have their plain and ordinary meaning as understood in light of the present specification, and refer to humans or non-human mammals such as dogs, cats, mice, rats, cows, sheep, pigs, goats, non-human primates, birds such as chickens, other vertebrates, or invertebrates. The term "mammal" is used in its normal biological sense. Thus, "mammal" specifically includes, but is not limited to, primates, including simians (chimpanzees, apes, monkeys) and humans, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, and the like.

[0115] As used herein, the term "effective amount" or "effective dose" has its plain and ordinary meaning as understood in light of the present specification, and refers to the amount of a composition or compound described herein that produces an observable effect. The actual dosage of the active ingredient contained in the active composition of the subject matter disclosed herein can be modified so that an effective amount of the active composition or compound is administered to obtain the desired response in a particular subject and / or application. The selected dosage will depend on a variety of factors, including, but not limited to, the activity of the composition, formulation, route of administration, combination with other drugs or treatments, the severity of the condition being treated, and the health and medical history of the subject being treated. In some embodiments, a minimum dose is administered, and the dose can be increased to the minimum effective amount without dose-limiting toxicity. Determination and adjustment of the effective dose, as well as evaluation of when and how to make such adjustments, are contemplated herein.

[0116] As used herein, the terms "function" and "functional" have their plain and ordinary meaning as understood in the context of this specification, and refer to a biological function, an enzymatic function or a therapeutic function.

[0117] As used herein, the term "inhibit" has its plain and ordinary meaning as understood in the context of the present specification and may mean to reduce or inhibit biological activity. The degree of reduction may be expressed as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, or about these values, at least these values, at least about these values, less than these values, less than these values, or within a range of values ​​bounded by any two of these values. As used herein, the term "delay" has its plain and ordinary meaning as understood in the context of the present specification and means to slow the progression of a biological event or to delay the occurrence of the event from the expected time. The degree of delay may be expressed as 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, about these values, at least these values, at least about these values, less than these values, less than these values, or within a range bounded by any two of these values. The terms "suppress" and "delay" do not necessarily mean 100% suppression or delay, but may be partial suppression or delay.

[0118] As used herein, the term "isolated" has its plain and ordinary meaning as understood in the context of the present specification and refers to (1) a substance and / or entity that is separated from at least some of the components that originally accompanied it when it was produced (in a natural and / or experimental environment) and / or (2) a substance and / or entity that is produced, prepared and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from other components that originally accompanied them by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, substantially 100% or 100%, about, at least about, at least about, at most, at most, at most, or ranges including and / or spanning these values. In some embodiments, the purity of an isolated material is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, substantially 100% or 100%, or is about, at least about, at least about, at most, at most, or ranges including and / or spanning these values. As used herein, an "isolated" material may be a "pure" (e.g., substantially free of other components) material. As used herein, the term "isolated cell" may refer to a cell that is not contained in a multicellular organism or tissue.

[0119] As used herein, "in vivo" has its plain and ordinary meaning as understood in the context of this specification, meaning that a procedure is performed within the body of a living organism, typically an animal, a mammal, including a human, or a plant, as opposed to a tissue extract or a dead organism.

[0120] As used herein, "ex vivo" has its plain and ordinary meaning as understood in the context of this specification, meaning performing any procedure outside of a living organism with minimal modification of the natural state.

[0121] As used herein, "in vitro" has its plain and ordinary meaning as understood in the context of this specification, and means performing some procedure in a condition other than the biological condition, for example, in a petri dish or test tube.

[0122] As used herein, the term "nucleic acid" or "nucleic acid molecule" has its plain and ordinary meaning as understood in the context of the present specification and refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, nucleic acids or molecules naturally occurring in cells, fragments obtained by polymerase chain reaction (PCR), and fragments obtained by ligation, cleavage, endonuclease action, and exonuclease action. Nucleic acid molecules may be composed of natural nucleotide monomers (such as DNA or RNA), analogs of natural nucleotides (e.g., enantiomers of natural nucleotides), or combinations thereof. Modified nucleotides may have modifications in the sugar moiety and / or the pyrimidine or purine base moiety. Modifications in the sugar moiety include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, or azide groups, and the sugar moiety may be etherified or esterified. Additionally, the entire sugar moiety may be replaced with conformationally or electronically similar structures, such as, for example, azasugars and carbocyclic sugar analogs. Modified base moieties include alkylated purines, alkylated pyrimidines, acylated purines, acylated pyrimidines, and other known heterocyclic substituents. Nucleic acid monomers can be linked by phosphodiester bonds or similar bonds. Linkages similar to phosphodiester bonds include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, and phosphoroamidate bonds. "Nucleic acid molecule" also includes so-called "peptide nucleic acids." Peptide nucleic acids contain natural or modified nucleobases attached to a polyamide backbone. Nucleic acids can be single-stranded or double-stranded. "Oligonucleotide" is used interchangeably with nucleic acid to mean double-stranded or single-stranded DNA or RNA.The nucleic acid may be contained in a nucleic acid vector or construct (e.g., a plasmid, a virus, a retrovirus, a lentivirus, a bacteriophage, a cosmid, a fosmid, a phagemid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC) or a human artificial chromosome (HAC)) that is capable of amplifying and / or expressing the nucleic acid in various biological systems. Typically, the nucleic acid vector or construct also contains elements such as, but not limited to, a promoter, an enhancer, a terminator, an inducer, a ribosome binding site, a translation initiation site, a start codon, a stop codon, a polyadenylation signal, an origin of replication, a cloning site, a multiple cloning site, a restriction enzyme site, an epitope, a reporter gene, a selection marker, an antibiotic selection marker, a target sequence, a peptide purification tag or an accessory gene, or any combination thereof.

[0123] A nucleic acid or a nucleic acid molecule may contain one or more sequences encoding a plurality of different peptides, polypeptides, or proteins. These one or more sequences may be adjacently linked within a single nucleic acid or a nucleic acid molecule, or may be linked via another nucleic acid. The other nucleic acid may be, for example, a linker, a repeat sequence, a restriction enzyme site, or a 1-base length, 2-base length, 3-base length, 4-base length, 5-base length, 6-base length, 7-base length, 8-base length, 9-base length, 10-base length, 11-base length, 12-base length, 13-base length, 14-base length, 15-base length, 16-base length, 17-base length, 18-base length, 19-base length, 20-base length, 25-base length, 30-base length, 35-base length, 40-base length, 45-base length, 50-base length, 55-base length, Examples of such sequences include sequences of 60 bases, 65 bases, 70 bases, 75 bases, 80 bases, 85 bases, 90 bases, 95 bases, 100 bases, 150 bases, 200 bases, or 300 bases in length, sequences of about these lengths, sequences of at least these lengths, sequences of at least these lengths, sequences of less than these lengths, sequences of less than these lengths, or sequences of lengths within a range of lengths with any two of these values ​​as upper and lower limits. In this specification, the term "downstream" in relation to a nucleic acid has a plain and ordinary meaning as understood in the light of this specification, and means a sequence located on the 3'-end side (rear) of a certain sequence on the strand (sense strand) containing the coding sequence when the nucleic acid is double-stranded. In this specification, the term "upstream" in relation to a nucleic acid has a plain and ordinary meaning as understood in the light of this specification, and means a sequence located on the 5'-end side (forward) of a certain sequence on the strand (sense strand) containing the coding sequence when the nucleic acid is double-stranded. As used herein, the term "grouped" with respect to nucleic acids has its plain and ordinary meaning as understood in the context of this specification, and refers to two or more sequences that are in close proximity, e.g., two or more sequences that are directly linked or linked via another nucleic acid in between.The other nucleic acid may be, for example, a linker, a repeat sequence, or a restriction enzyme site, or a 1-base length, 2-base length, 3-base length, 4-base length, 5-base length, 6-base length, 7-base length, 8-base length, 9-base length, 10-base length, 11-base length, 12-base length, 13-base length, 14-base length, 15-base length, 16-base length, 17-base length, 18-base length, 19-base length, 20-base length, 25-base length, 30-base length, 35-base length, 40-base length, 45-base length, 50-base length, 55-base length, Examples of such sequences include sequences that are 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, sequences of approximately these lengths, sequences of at least these lengths, sequences of at least these lengths, sequences of less than these lengths, sequences of less than these lengths, or sequences of a length within a range of any two of these values. Note that the intervening nucleic acid sequences do not typically code for functional or catalytic polypeptides, proteins, or protein domains.

[0124] The nucleic acid described herein comprises nucleic acid base. The basic base, in other words, the standard base, the natural base or the unmodified base, is adenine, cytosine, guanine, thymine and uracil. Other nucleic acid bases include, but are not limited to, purines, pyrimidines, modified nucleic acid bases, 5-methylcytosine, pseudouridine, dihydrouridine, inosine, 7-methylguanosine, hypoxanthine, xanthine, 5,6-dihydrouracil, 5-hydroxymethylcytosine, 5-bromouracil, isoguanine, isocytosine, aminoallyl base, dye-labeled base, fluorescent-labeled base or biotin-labeled base.

[0125] As used herein, "peptide", "polypeptide" and "protein" have their plain and common meaning as understood in the context of this specification, and refer to a macromolecule formed by linking multiple amino acids by peptide bonds. Numerous functions of peptides, polypeptides and proteins are known in the art, including, but not limited to, enzymatic, structural, transport, defensive, hormonal or signal transduction functions. Peptides, polypeptides and proteins are often biologically produced by ribosomal complexes using nucleic acids as templates, but are not limited to this method and can also be produced by chemical synthesis. Mutations can be added to peptides, polypeptides or proteins by genetically engineering template nucleic acids, including substitutions, deletions, truncations, additions, duplications, or fusion of two or more peptides, polypeptides or proteins. When two or more peptides, polypeptides or proteins are fused, the two or more peptides, polypeptides or proteins can be linked adjacent to each other in one molecule, or can be linked with another amino acid in between. The additional amino acids may be, for example, a linker, a repeat sequence, an epitope or a tag, or a 1-base length, a 2-base length, a 3-base length, a 4-base length, a 5-base length, a 6-base length, a 7-base length, a 8-base length, a 9-base length, a 10-base length, a 11-base length, a 12-base length, a 13-base length, a 14-base length, a 15-base length, a 16-base length, a 17-base length, a 18-base length, a 19-base length, a 20-base length, a 25-base length, a 30-base length, a 35-base length, a 40-base length, a 45-base length, a 50-base length, a 55-base length, Examples of sequences that can be used include sequences that are 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, sequences that are approximately these lengths, sequences that are at least these lengths, sequences that are at least approximately these lengths, sequences that are shorter than these lengths, sequences that are approximately shorter than these lengths, or sequences that are within a range of lengths that are upper and lower limits of any two of these values. As used herein, the term "downstream" in reference to a polypeptide has its plain and ordinary meaning as understood in light of the present specification, and refers to a sequence that is C-terminal (rear) to a sequence.As used herein, the term "upstream" with respect to a polypeptide has its plain and ordinary meaning as understood in the context of this specification, meaning a sequence that is N-terminal (in front of) another sequence.

[0126] As used herein, "% w / w" or "% wt / wt" has its plain and ordinary meaning as understood in the context of this specification, which is the ratio of the weight of a component or agent to the total weight of a composition of the invention, multiplied by 100. As used herein, "% v / v" or "% vol / vol" has its plain and ordinary meaning as understood in the context of this specification, which is the ratio of the liquid volume of a compound, substance, component or agent to the total liquid volume of a composition of the invention, multiplied by 100.

[0127] As used herein, the term "urinary exfoliated cells" refers to a small number of urothelial and other cells associated with the urinary tract that may be shed during urine excretion, including potentially cancerous cells. By analyzing the shed cells (e.g., cancer cells, red blood cells, white blood cells, or bacteria) in a urine specimen, it is possible to non-invasively assess the health status of an individual. However, the concentration of the urinary exfoliated cells may be very low, and it may be desirable and / or necessary to concentrate them. This can be done by compressing the cellular components by centrifugation into a cell pellet ("urinary sediment cells"), which can then be resuspended and used for further processing.

[0128] Specific cellular target substances in a specimen can be detected and stained. The specific cellular target substances can be any biological component, including but not limited to proteins or nucleic acids. Two approaches are immunohistochemistry (immunocytochemistry), which uses antibodies to detect proteins (or other epitopes to which antibodies can bind), and in situ hybridization (fluorescence, chromogenic, etc.), which uses nucleic acid probes to hybridize and detect nucleic acids such as DNA or RNA. In some embodiments, immunocytochemistry and chromogenic in situ hybridization methods specific for bladder cancer-specific biomarkers are disclosed herein. Chromogenic methods, in which a chromogenic substrate is enzymatically converted to a colored precipitate, can be used, particularly for ease of detection using conventional optical microscopy. However, it is envisioned that other biomarker-specific stains or other biomarkers may be used instead by those skilled in the art.

[0129] Typically, immunocytochemistry may be performed using glass slides. The cell or tissue specimen on the slide may be incubated with a primary antibody specific to the target protein. After thorough washing to remove the primary antibody, a chromogenic enzyme-labeled secondary antibody may be added to the slide and incubated. The corresponding chromogenic substrate may then be added to the slide, resulting in a colored precipitate catalyzed by the chromogenic enzyme bound to the secondary antibody, giving a positive signal. The signal on the slide may be evaluated by light microscopy.

[0130] Typically, chromogenic in situ hybridization may be performed using glass slides. Cell or tissue samples on the slides may be incubated with specific nucleic acid probes that hybridize with the target nucleic acid. After thorough washing to remove the nucleic acid probes, chromogenic enzyme-labeled secondary reagents may be added to the slides in sequence and incubated. Then, the corresponding chromogenic substrates may be added to the slides, which may produce a color precipitate catalyzed by the chromogenic enzyme bound to the secondary reagents, resulting in a positive signal. The signals on the slides may be evaluated by light microscopy.

[0131] Immunocytochemistry may use protein antibodies, whereas chromogenic in situ hybridization may use nucleic acid probes as the primary reagent. Signal generation and evaluation of these two methods may yield similar results.

[0132] As used herein, the term "cytomorphological stain" refers to a staining method that generally stains cells or other biological material with a dye or mixture of dyes (as opposed to immunocytochemistry or chromogenic in situ hybridization staining methods, which target specific components). Cytomorphological stains are often used to enhance the contrast of cells in conventional light microscopy and to provide discernible details about cell morphology for cytopathological assessment. Examples of cytomorphological stains include Diff-Quick, Papanicolaou, Wright-Giemsa, and Hematoxylin / Eosin. Derivatives and modifications of known cytomorphological stains are also contemplated for use due to specific desired properties, either using the same or different chemical dyes to provide the stain.

[0133] In at least some of the above embodiments, where technically feasible, one or more components used in any embodiment may be substituted for components in another embodiment. Those skilled in the art will appreciate that various omissions, additions and modifications, other than those described above, may be made to the methods and structures described herein without departing from the scope of the claimed subject matter. All such modifications and variations are within the scope of the subject matter defined by the appended claims.

[0134] With respect to the use of substantially all plural and / or singular terms herein, those of ordinary skill in the art may interpret the plural as singular and / or the singular as plural, as appropriate to the context and / or application. For the sake of clarity, such various singular and plural interchanges may be expressly set forth herein.

[0135] "Eg" means "for example," and is therefore understood to be non-limiting.

[0136] As will be appreciated by those of skill in the art, generally, the terms used herein, and particularly in the appended claims (e.g., the body of the appended claims), are intended to be "open" terms throughout (e.g., the term "including" is to be interpreted as "including, but not limited to," the term "having" is to be interpreted as "having at least," the term "including" is to be interpreted as "including, but not limited to," etc.). Furthermore, those of skill in the art will appreciate that where a specific number is intended in a claim recitation, such intent will be expressly set forth in the claim, and that otherwise no such intent exists. To be more specific, for example, the appended claims may use prefaces such as "at least one" or "one or more" in the claim recitation. However, the use of such a preamble does not limit a particular claim containing a statement using the indefinite article "a" to embodiments containing only that statement, even if a claim contains a preamble such as "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a and / or an" is interpreted to mean "at least one" or "one or more"). This also applies to claims using definite articles. In addition, even if a specific number is explicitly recited in a claim, one of ordinary skill in the art would understand that the recited number is a minimum number (e.g., "two" without a modifier means "at least two" or "two or more"). Furthermore, when an idiomatic expression such as "at least one of A, B, and C" is used, it is intended to be interpreted in the sense that a person of ordinary skill in the art would normally understand the idiomatic expression (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, a system having only A, a system having only B, a system having only C, a system having A and B, a system having A and C, a system having B and C, and / or a system having A, B, and C, etc.).In addition, when an idiomatic expression such as "at least one of A, B, or C" is used, it is intended to be interpreted in a way that a person skilled in the art would normally understand the idiomatic expression (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, a system having only A, a system having only B, a system having only C, a system having A and B, a system having A and C, a system having B and C, and / or a system having A, B, and C). Furthermore, a person skilled in the art would understand that disjunctive words and / or disjunctive phrases for presenting two or more options contemplate the possibility of including one of the terms listed as options, either of the terms, or both of the terms in the specification, claims, or drawings. For example, the expression "A or B" is understood to include the possibility of "A" or "B", or "A and B".

[0137] Furthermore, when features or aspects of the disclosure are described in Markush format, it will be understood by those skilled in the art that the disclosure also be described in terms of any individual element or subgroup of elements in the Markush format.

[0138] It will be understood by those skilled in the art that all ranges described herein, for any purpose, including but not limited to providing a detailed description, are intended to encompass all possible subranges and combinations of subranges. Any of the ranges described above will be fully described and readily understood as being divisible into at least two, three, four, five, ten, etc. parts. For example, any of the ranges described herein can be readily divided into three parts, such as upper, middle, lower, etc., but are not limited thereto. Additionally, any of the terms "less than", "at least", "greater than", "less than" and the like will be understood by those skilled in the art to include the numerical values ​​described and also represent ranges that can be divided into subranges as described herein. Additionally, any of the ranges described herein will be understood by those skilled in the art to include individual elements. Thus, for example, a group having 1 to 3 elements represents a group having one element, a group having two elements, or a group having three elements. Similarly, a group having 1 to 5 elements refers to a group having 1 element, a group having 2 elements, a group having 3 elements, a group having 4 elements, or a group having 5 elements, and so forth.

[0139] While various aspects and embodiments have been disclosed herein, those skilled in the art will readily appreciate that other aspects and embodiments are possible. The various aspects and embodiments disclosed herein are intended to be illustrative of the invention and are not intended to limit the invention in any way, the scope and spirit of the invention being indicated by the following claims.

[0140] All references cited herein, including but not limited to published patent applications, unpublished patent applications, patents, and journal articles, are incorporated herein by reference for their specific disclosures or in their entirety as cited herein and made a part hereof. In the event that a publication, patent, or patent application incorporated by reference conflicts with the disclosure of this specification, the disclosure of this specification will govern and / or control over any such conflicting matter.

[0141] Cytopathological multiplex staining equipment Disclosed herein is an apparatus for performing multiple cytopathological staining. Generally, the apparatus can use a single staining chamber to process cells from a biological specimen through multiple staining and washing steps, and a final collection step to collect the cells for further manipulation (e.g., mounting on a microscope slide for visualization). Such an embodiment allows for multiple staining in a "one-pot" manner, eliminating the need for separate operations such as centrifugation for cell collection and removal of used staining and washing reagents. The "one-pot" method may be advantageous for biological specimens with a low proportion of atypical cells and / or a low total number of cells, such as in the case of urine sediment cells for the diagnosis of bladder cancer. Furthermore, the apparatus according to the present disclosure may automate the multiple staining and washing steps, which may reduce variability in specimen processing and improve the reliability of cytopathological examination.

[0142] In some embodiments, the device of the present invention comprises: a) a staining vessel capable of containing aqueous cell culture medium, staining reagents, washing reagents and / or slide mounting medium, as well as cells from a biological specimen obtained from a patient; b) an extraction vessel configured to receive the used aqueous cell culture medium, the used staining reagent, and / or the used washing reagent, and configured to allow the used aqueous cell culture medium, the used staining reagent, and / or the used washing reagent to be removed; Includes. In some embodiments, the staining vessel includes one or more aqueous media, staining reagents, washing reagents, and / or slide mounting medium. In some embodiments, the cells may be resuspended in a series of aqueous cell media, staining reagents, washing reagents, and / or slide mounting medium. In some embodiments, the extraction vessel may be capable of and configured to accommodate a series of used aqueous cell media, used staining reagents, and / or used washing reagents. In some embodiments, the staining vessel and the extraction vessel may be operably separated via a semi-permeable membrane that is permeable to the aqueous cell media, staining reagents, washing reagents, and / or mounting medium, but not to the cells, thereby retaining the cells in the staining vessel. In some embodiments, the staining vessel is configured to provide stained cells by alternating between staining and washing steps. In some embodiments, the cells are provided by passing through a series of alternating staining and washing steps in the staining vessel. In some embodiments, the staining reagents of each staining step and the washing reagents of each washing step in the staining vessel can be extracted into the extraction vessel when used. In some embodiments, the used staining reagent and the used washing reagent can be removed from the extraction vessel.

[0143] In some embodiments, the semipermeable membrane comprises pores that allow the passage of liquid and sufficiently small molecules, but not cells, and in some embodiments, each pore may have a diameter of 0.05 μm, 0.1 μm, 0.2 μm, 0.22 μm, 0.3 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 3 μm, 5 μm, or 10 μm, or a range of diameters bounded by any two of these values.

[0144] In some embodiments, the staining step performed in the device of the present invention comprises staining the cells with a cytomorphological stain, an immunocytochemical stain, or a chromogenic in situ hybridization stain, or any combination thereof. In some embodiments, the cytomorphological stain comprises a Diff-Quick stain, a Papanicolaou stain, a Wright-Giemsa stain, a Hematoxylin / Eosin stain, or any derivative, combination, or variation thereof.

[0145] In some embodiments, the staining reagents of each staining step and the washing reagents of each washing step in the staining vessel can be extracted into the extraction vessel by passive diffusion, or the staining reagents and washing reagents in the staining vessel can be extracted through the semipermeable membrane into the extraction vessel by applying negative pressure to the extraction vessel. In some embodiments, the device of the present invention further comprises an extraction pump. In some embodiments, the extraction pump applies a relative negative pressure to the extraction vessel compared to the staining vessel. In some embodiments, the staining vessel and the extraction vessel can be physically separated or blocked by a shutter, which may prevent, inhibit and / or reduce the movement of the aqueous cell medium, the staining reagent and / or the washing reagent between the staining vessel and the extraction vessel through the semipermeable membrane.

[0146] In some embodiments, the device of the present invention further comprises a reagent source that may be capable of providing aqueous cell medium, staining reagents, washing reagents and / or slide mounting medium to the staining vessel or that is configured to provide these media or reagents to the staining vessel. In some embodiments, the device of the present invention further comprises a reagent pump. In some embodiments, the reagent source may be operably coupled to a reagent pump configured to provide a mechanical force that causes the aqueous cell medium, staining reagents, washing reagents and / or slide mounting medium to be provided from the reagent source. In some embodiments, the aqueous cell medium comprises cells for performing an initial operation of the device of the present invention.

[0147] In some embodiments, the device further comprises a specimen collection channel that may be capable of collecting stained cells, e.g., for mounting, or configured to collect stained cells. In some embodiments, after final manipulation of the device, the stained cells may be contained in a slide mounting medium. In some embodiments, the stained cells may be collected manually (e.g., with a pipette, syringe, etc.) or may be collected by applying negative pressure through the specimen collection channel. In some embodiments, the specimen collection channel may utilize negative pressure to collect stained cells.

[0148] In some embodiments, the devices of the present invention may be modified from a cytological specimen and slide preparation system, such as the ThinPrep 2000 system.

[0149] One non-limiting embodiment of an apparatus for performing multiplex cytopathological staining in a "vertical" configuration is shown in Figures 1A-C.

[0150] 1A shows an exemplary apparatus, comprising a staining vessel 100, which may contain cells 101, isolated and / or purified from a biological specimen obtained from a patient, in suspension in a suitable liquid medium 103.

[0151] In some embodiments, the staining vessel 100 may be a vessel with an interior volume and an opening suitable for containing the cells 101 and the liquid medium 103. In some embodiments, the staining vessel 100 may comprise a bio-inert material such as plastic, glass, quartz, steel, titanium, ceramic, alumina, zirconia, or other materials commonly used in cell culture. In some embodiments, the staining vessel 100 may be a tube, a cylindrical tube, a rectangular tube, a cuvette, a dish, a plate, or other vessel usable in cell culture. In some embodiments, the staining vessel 100 may be optically transparent or clear, or configured to allow visualization of the contents within the vessel. In some embodiments, the staining vessel 100 may be fitted with a holder that can be attached to the staining vessel 100 to keep the staining vessel 100 fixed in place.

[0152] In some embodiments, cells 101 isolated and / or purified from a biological specimen obtained from a patient may be fixed and / or permeabilized prior to placement in the staining container 100. In some embodiments, cells 101 may be fixed and / or permeabilized in the staining container 100 prior to operating the exemplary apparatus. In some embodiments, cells 101 may be fixed and / or permeabilized in the staining container 100 by operating the exemplary apparatus.

[0153] As shown in FIG. 1A, the exemplary device further includes an extraction vessel 102 with two open ends, one of which may be fitted with a semi-permeable membrane 104 that may be permeable to liquid medium 103 (and small molecules such as proteins, antibodies, nucleic acids, small molecule staining dyes, etc.) but not to cells 101. In some embodiments, the extraction vessel 102 may be placed within the interior volume of the staining vessel 100. In some embodiments, the extraction vessel 102 may comprise a bio-inert material such as plastic, glass, quartz, steel, titanium, ceramic, alumina, zirconia, or other materials commonly used in cell culture. In some embodiments, the extraction vessel 102 may be a tube, a cylindrical tube, a square tube, or other shape that may be placed within the interior volume of the staining vessel 100. In some embodiments, the extraction vessel 102 may be optically transparent or clear, or may be configured to allow visualization of the contents in the vessel. In some embodiments, the other of the two open ends (i.e., the open end not fitted with the semi-permeable membrane 104) may be operatively connected to an extraction pump 115 (e.g., an air pump) capable of applying a negative pressure 107, which may extract atmospheric gas or liquid medium 103 from the dyeing vessel 100 through the semi-permeable membrane 104. In some embodiments, the extraction pump 115 may further apply a positive pressure, which may expel the contents in the extraction vessel 102 through the semi-permeable membrane 104 to the dyeing vessel 100 or another location (e.g., a waste vessel). In some embodiments, the extraction pump 115 may not be provided and the contents in the extraction vessel 102 may be manually manipulated.

[0154] In some embodiments, the extraction vessel 102 can be manipulated to move its position relative to the staining vessel 100. In some embodiments, 1) the extraction vessel 102 can be moved without immersing the extraction vessel 102 and the semipermeable membrane 104 in the liquid medium 103, or 2) the extraction vessel 102 can be moved while the semipermeable membrane 104 and at least a portion of the extraction vessel 102 closest to the semipermeable membrane 104 (i.e., the portion of the extraction vessel 102 to which the semipermeable membrane 104 may be attached) are immersed in the liquid medium 103. In some embodiments, the extraction vessel 102 can be moved without the entire extraction vessel 102 and the semipermeable membrane 104 being within the interior volume of the staining vessel 101. In some embodiments, the extraction vessel 102 can be manipulated manually or with a motor.

[0155] In some embodiments, the semipermeable membrane 104 comprises pores that allow liquid and sufficiently small molecules to pass through, but do not allow cells to pass through. In some embodiments, the pores have a diameter of 0.05 μm, 0.1 μm, 0.2 μm, 0.22 μm, 0.3 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 3 μm, 5 μm, or 10 μm, or a range of diameters bounded by any two of these values. The pore size of the semipermeable membrane 104 can be adjusted depending on the cells 101 to be treated. In some embodiments, the semipermeable membrane 104 may comprise cellulose acetate (CA), fiberglass, nylon, polyethersulfone (PES), polypropylene (PP), polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), regenerated cellulose (RC), or other materials commonly used in semipermeable membranes and which may be bioinert.

[0156] As shown in FIG. 1A, the exemplary device may include a reagent supply or source 105. The reagent supply or source 105 may include a conduit adapted to supply liquid reagents 112, such as staining dyes and washing reagents, to the staining vessel 100. In some embodiments, the reagent supply may be manually operated, such as a pipette. In some embodiments, the reagent supply 105 may be operably connected to a reagent pump 113 capable of providing the liquid reagents 112 and providing mechanical force to drive the liquid reagents through the reagent supply 105 to the staining vessel 100. In some embodiments, the reagent supply 105 and / or the reagent pump 113 may be stationary or not, and all supply of reagents 112 may be performed manually, such as with a pipette. Addition of reagents 112 via the reagent supply 105 (or performed manually) typically marks the start of a respective staining or washing step. In some embodiments, the reagent supply 105 may be operable to move its position relative to the staining vessel 100. In some embodiments, a reagent supply device 105 can be utilized to deliver an initial suspension of cells 101 to the staining container 100 to initiate operation of the exemplary apparatus (in this case, at the start of operation of the exemplary apparatus, the initial suspension of cells 101 may be indicated by the reference numeral 112).

[0157] As disclosed in the methods provided herein, the schematic diagram of FIG. 1A may represent an initial step of loading cells 101 into a staining container 100, or each of one or more staining steps that may involve staining (e.g., cytomorphological staining, immunocytochemical staining, or chromogenic in situ hybridization staining) or washing of cells. In this case, liquid medium 103 represents liquid reagents including components for each staining step, such as small molecule staining dyes, antibodies, nucleic acids, chromogenic reagents, or washing reagents for washing cells after each staining step. In some embodiments, these liquid reagents and / or washing reagents may be delivered via a reagent supply device 105 or may be supplied manually.

[0158] 1B shows an intermediate position of the exemplary device illustrated in FIG. 1A, illustrating an extraction step, which may occur following an initial cell input step and is performed after each staining step and after each washing step to remove the starting liquid medium in which the cells may be suspended (and which may contain fixative and / or permeabilization reagents). During this extraction step, the extraction vessel 102 may be manipulated while the semi-permeable membrane 104 and at least a portion of the extraction vessel 102 are immersed in the liquid medium 103 containing the cells 101. In some embodiments, the liquid medium 103 and sufficiently small molecules (such as components used for staining or washing) may flow from the staining vessel 100 to the extraction vessel 102 by passive diffusion through the semi-permeable membrane 104 while the cells 101 remain in the staining vessel 100. In some embodiments, negative pressure 107 in the extraction vessel 102 allows the liquid medium 103 and sufficiently small molecules (such as components used for staining or washing) to flow from the staining vessel 100 to the extraction vessel 102 through the semipermeable membrane 104, while leaving the cells 101 in the staining vessel 100. In some embodiments, the negative pressure 107 may be applied by an extraction pump 115, which may or may not be installed, and the contents in the extraction vessel 102 may be manually operated. The extracted liquid 106 in the extraction vessel 102 may be removed as waste. In some embodiments, after the extracted liquid 106 is removed, the cells 101 retained by the semipermeable membrane 104 may be moved back into homogenous suspension by positive gas pressure in the extraction vessel 102. In FIG. 1B, the reagent supply 105, the reagent 112, and the reagent pump 113 are not shown. However, this does not necessarily imply that the reagent supply apparatus 105, the reagent 112, and the reagent pump 113 need to be removed or moved during the steps illustrated in FIG. 1B, as these components may or may not be present during operation of the apparatus at any step.

[0159] The depth at which the extraction vessel 102 may be placed within the staining vessel 100 may be adjusted, but it may be desirable to position the extraction vessel 102 with the semi-permeable membrane 104 as close as possible to the bottom of the staining vessel 100 to extract as much of the liquid medium 103 as possible, while leaving enough space for the liquid medium 103 and cells 101 to flow freely without excessive shear forces. The liquid medium 103 remaining after this step may be diluted in an appropriate washing step to remove components not required for the next step.

[0160] As disclosed in the methods provided herein, the schematic diagram of FIG. 1B may be representative of one or more staining and washing steps, with liquid medium 103 representing liquid reagents containing components for each staining step, such as small molecule staining dyes, antibodies, nucleic acids, color-developing reagents, or a washing reagent for washing cells after each staining step.

[0161] FIG. 1C shows a schematic diagram illustrating the final operation step of the exemplary device specifically shown in FIGS. 1A-B. At this stage, each staining step and each washing step have already been performed, and the stained cells 110 have been sufficiently stained and washed as desired. The stained cells may be contained in the staining container 100 throughout the staining and washing steps, and may be resuspended in the final liquid medium 108. In some embodiments, the final liquid medium 108 may be a standard aqueous medium (e.g., phosphate buffered saline or other suitable buffer medium) or an aqueous medium (e.g., a mounting medium) that may be optimized for slide mounting, visualization, and / or staining stability. The final stained cells 110 may be collected from the staining container and mounted on a slide for diagnostic testing. In some embodiments, the slide with the stained cells 110 mounted thereon may be any of the slide assemblies disclosed herein. The stained cells 110 may be collected manually, such as by using a pipette, or may be collected using a specimen collection channel 109. In some embodiments, the specimen collection channel 109 can collect the final liquid medium 108 and the stained cells 110 contained therein by negative pressure. In some embodiments, the specimen collection channel can be operatively connected to another device for automated encapsulation of a slide for visualization, such as any of the slide assemblies disclosed herein. In FIG. 1C, the reagent supply device 105, the reagent 112, and the reagent pump 113 are not shown. However, this does not necessarily imply that the reagent supply device 105, the reagent 112, and the reagent pump 113 need to be removed or moved during the steps shown in FIG. 1C, and these components may or may not be present during operation of the device at any step. This also applies to the extraction vessel 102 and the semipermeable membrane 104. Although these components are not shown in FIG. 1C, this does not necessarily imply that these components need to be removed during operation of the device, and the extraction vessel 102 and semi-permeable membrane 104 may or may not be present, or may be located in another location to aid in the operational steps of FIG. 1C, etc.

[0162] Another non-limiting embodiment of an apparatus for performing multiplex cytopathological staining in a "horizontal" configuration is shown in Figures 1D-F.

[0163] The initial setup of an exemplary apparatus is shown in Figure ID. A staining vessel 100 may be provided, which may contain cells 101 isolated and / or purified from a biological specimen obtained from a patient in suspension in a suitable liquid medium 103.

[0164] In some embodiments, the staining vessel 100 may be any vessel with an interior volume and opening suitable for containing the cells 101 and the liquid medium 103. In some embodiments, the staining vessel 100 may comprise a bio-inert material such as plastic, glass, quartz, steel, titanium, ceramic, alumina, zirconia, or other materials commonly used in cell culture. In some embodiments, the staining vessel 100 may be a tube, a cylindrical tube, a rectangular tube, a cuvette, a dish, a plate, or other vessel usable in cell culture. In some embodiments, the staining vessel 100 may be optically transparent or clear, or configured to allow visualization of the contents of the vessel. In some embodiments, the staining vessel 100 may be fitted with a holder that can be attached to the staining vessel 100 to keep the staining vessel 100 fixed in place.

[0165] In some embodiments, cells 101 isolated and / or purified from a biological specimen obtained from a patient may be fixed and / or permeabilized prior to placement in the staining container 100. In some embodiments, cells 101 may be fixed and / or permeabilized in the staining container 100 prior to operating the exemplary apparatus. In some embodiments, cells 101 may be fixed and / or permeabilized in the staining container 100 by operating the exemplary apparatus.

[0166] As shown in FIG. 1D, the exemplary device may include an extraction vessel 102 that may be operably connected to the staining vessel 100, but may be separated from the staining vessel 100 via a semi-permeable membrane 104 that may be permeable to the liquid medium 103 (and small molecules such as proteins, antibodies, nucleic acids, small molecule staining dyes, etc.) but not to the cells 101, and may be separated from the staining vessel 100 via a shutter 111 adjacent to the semi-permeable membrane 104 that is positioned to block flow between the staining vessel 100 and the extraction vessel 102 through the semi-permeable membrane 104. In some embodiments, the extraction vessel 102 and / or the shutter 111 may comprise a bioinert material, such as plastic, glass, quartz, steel, titanium, ceramic, alumina, zirconia, or other materials commonly used in cell culture. In some embodiments, the extraction vessel 102 may be a tube, a cylindrical tube, a square tube, or any other shape that can be arranged to be operatively connected to the staining vessel 100, such as adjacent to or surrounded by the staining vessel 100, so long as the semi-permeable membrane 104 and the shutter 111 provide separation between the staining vessel 100 and the extraction vessel 102. In some embodiments, the extraction vessel 102 and / or the shutter 111 may be optically transparent to allow observation of the contents of the vessel. In some embodiments, the extraction vessel 102 may be sealed except for its connection to the staining vessel 100, which is separated by the semi-permeable membrane 104 (which may be completely sealed by the shutter 111 and the extraction opening 114). In some embodiments, the extraction opening 114 of the extraction vessel 102 may be appropriately sized to allow removal of its contents, such as with a syringe or pipette, or manually. In some embodiments, the extraction opening 114 of the extraction vessel 102 may be operably connected to an extraction pump 115 capable of applying a negative pressure 107 to remove the contents of the extraction vessel 102.In this case, the extraction opening 114 may have a flow path extending to or near the bottom of the extraction vessel 102 to allow liquid to be extracted from the extraction vessel 102. In some embodiments, the extraction pump 115 may further apply a positive pressure, which may expel the contents in the extraction vessel 102 into the dyeing vessel 100 when the shutter 111 may be open. In some embodiments, the extraction pump 115 may not be provided and the contents in the extraction vessel 102 may be manually manipulated.

[0167] In some embodiments, the semipermeable membrane 104 comprises pores that allow liquid and sufficiently small molecules to pass through, but do not allow cells to pass through. In some embodiments, the pores have a diameter of 0.05 μm, 0.1 μm, 0.2 μm, 0.22 μm, 0.3 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 3 μm, 5 μm, or 10 μm, or a range of diameters bounded by any two of these values. The pore size of the semipermeable membrane 104 can be adjusted depending on the cells 101 to be treated. In some embodiments, the semipermeable membrane 104 may comprise cellulose acetate (CA), fiberglass, nylon, polyethersulfone (PES), polypropylene (PP), polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), regenerated cellulose (RC), or other materials commonly used in semipermeable membranes and which may be bioinert.

[0168] As shown in FIG. 1D, the exemplary device may include a reagent supply device 105. The reagent supply device 105 may include a conduit adapted to supply liquid reagents 112, such as staining dyes and washing reagents, to the staining vessel 100. In some embodiments, the reagent supply device 105 may be manually operated, such as a pipette. In some embodiments, the reagent supply device 105 may be operably connected to a reagent pump 113 capable of providing the liquid reagents 112 and providing mechanical force to drive the liquid reagents through the reagent supply device 105 to the staining vessel 100. In some embodiments, the reagent supply device 105 and / or the reagent pump 113 may be stationary or not, and all supply of reagents 112 may be performed manually, such as with a pipette. Addition of reagents 112 via the reagent supply device 105 (or performed manually) typically marks the start of a respective staining or washing step. In some embodiments, the reagent supply device 105 may be operable to move its position relative to the staining vessel 100. In some embodiments, a reagent supply device 105 can be utilized to deliver an initial suspension of cells 101 to the staining container 100 to initiate operation of the exemplary apparatus (in this case, at the start of operation of the exemplary apparatus, the initial suspension of cells 101 may be indicated by the reference numeral 112).

[0169] As disclosed in the methods provided herein, the schematic diagram of FIG. 1D may represent an initial step of loading cells 101 into the staining container 100, or each of one or more staining steps that may involve staining (e.g., cytomorphological staining, immunocytochemical staining, or chromogenic in situ hybridization staining) or washing of cells. In this case, liquid medium 103 represents liquid reagents including components for each staining step, such as small molecule staining dyes, antibodies, nucleic acids, chromogenic reagents, or washing reagents for washing cells after each staining step. In some embodiments, these liquid reagents and / or washing reagents may be delivered via a reagent supply device 105 or may be supplied manually.

[0170] FIG. 1E shows an intermediate position of the exemplary device illustrated in FIG. 1D, illustrating the extraction step. This extraction step may be performed following the initial cell input step, and may be performed after each staining step and after each washing step to remove the starting liquid medium in which the cells may be suspended (and which may contain fixative and / or permeabilization reagents). In this extraction step, the shutter 111 (shown closed in FIG. 1D) may be opened, lifted, or otherwise removed to position the staining container 100 and the extraction container 102 separated only by the semipermeable membrane 104, allowing the transfer of liquid medium between the staining container 100 and the extraction container 102 through the semipermeable membrane 104. In some embodiments, the liquid medium 103 and sufficiently small molecules (such as components used for staining or washing) may pass from the staining container 100 to the extraction container 102 through the semipermeable membrane 104 by passive diffusion, while the cells 101 remain in the staining container 100. In some embodiments, negative pressure 107 in the extraction vessel 102 allows the liquid medium 103 and sufficiently small molecules (such as components used for staining or washing) to flow from the staining vessel 100 to the extraction vessel 102 through the semipermeable membrane 104, while leaving the cells 101 in the staining vessel 100. In some embodiments, the negative pressure 107 may be applied by an extraction pump 115. However, the extraction pump 115 may not be provided and the contents in the extraction vessel 102 may be manually manipulated. The extracted liquid 106 may be removed as waste, such as through an extraction opening 114. The liquid medium 103 remaining in the staining vessel 100 may be diluted in an appropriate washing step to remove components not required for the next step. In some embodiments, after removing the extracted liquid 106, the cells 101 retained by the semipermeable membrane 104 may be moved back into homogenous suspension by positive gas pressure in the extraction vessel 102. In FIG. 1E, the reagent supply 105, the reagents 112, and the reagent pump 113 are not shown.However, this does not necessarily imply that the reagent supply apparatus 105, the reagent 112, and the reagent pump 113 need to be removed or moved during the steps shown in FIG. 1E, as these components may or may not be present during operation of the apparatus at any step.

[0171] As disclosed in the methods provided herein, the schematic diagram of FIG. 1E may be representative of one or more staining and washing steps, with liquid medium 103 representing liquid reagents containing components for each staining step, such as small molecule staining dyes, antibodies, nucleic acids, color-developing reagents, or a washing reagent for washing the cells after each staining step.

[0172] FIG. 1F shows a schematic diagram illustrating the final operation step of the exemplary device specifically shown in FIGS. 1D-E. At this stage, each staining step and each washing step have already been performed, and the stained cells 110 have been sufficiently stained and washed as desired. The stained cells may be contained in the staining container 100 throughout the staining and washing steps, and may be resuspended in the final liquid medium 108. In some embodiments, the final liquid medium 108 may be a standard aqueous medium (e.g., phosphate buffered saline or other suitable buffer medium) or an aqueous medium (e.g., a mounting medium) that may be optimized for slide mounting, visualization, and / or staining stability. The final stained cells 110 may be collected from the staining container and mounted on a slide for diagnostic testing. In some embodiments, the slide in which the stained cells 110 may be mounted may be any of the slide assemblies disclosed herein. The collection of the stained cells 110 may be performed manually, such as with a pipette, or may be performed using a specimen collection channel 109. In some embodiments, the specimen collection channel 109 can collect the final liquid medium 108 and the stained cells 110 contained therein by negative pressure. In some embodiments, the specimen collection channel can be operatively connected to another device for automated encapsulation of a visualization slide, including any of the slide assemblies disclosed herein. In FIG. 1F, the reagent supply 105, the reagent 112, and the reagent pump 113 are not shown. However, this does not necessarily suggest that the reagent supply 105, the reagent 112, and the reagent pump 113 need to be removed or moved during the steps shown in FIG. 1F, and these components may or may not be present during operation of the device at any step.

[0173] Further disclosed herein is a method for multiplex cytopathological staining of cells from a biological specimen obtained from a patient using the apparatus disclosed herein, as exemplified in Figures 1A-F. In some embodiments, the staining step comprises staining the cells with a cytomorphological stain, an immunocytochemical stain, or a chromogenic in situ hybridization stain, or any combination thereof. In some embodiments, the cytomorphological stain comprises a Diff-Quick stain, a Papanicolaou stain, a Wright-Giemsa stain, a hematoxylin / eosin stain, or a derivative or variation thereof. In some embodiments, the cells comprise atypical cells. In some embodiments, the cells comprise cancer cells. In some embodiments, the cells comprise urinary sediment cells. In some embodiments, the urine sediment cells may stain for one or more bladder cancer specific biomarkers, which may be selected from the group consisting of S100P, p63, M344, LDQ10, 19A211, GATA-3, Ki-67, p16, Her-2, PD-L1, CTLA4, CK-17, CK-20, nmp-22, bladder tumor antigen (BTA), hTERT, and minichromosome maintenance protein 5 (MCM5).

[0174] In any of the embodiments provided herein, the patient may be a mammal. In some embodiments, the patient may be a human.

[0175] Cytopathology Slide Assembly Further disclosed herein is a pathology slide assembly including a pathology slide and a pathology cover slip. In some embodiments, the pathology slide and the pathology cover slip can be stacked together and sealed to define an interior space accessible through an inlet port and an outlet port of the pathology slide assembly. In some embodiments, a suspension of cells from a biological specimen obtained from a patient can be delivered to the interior space through the inlet port, and cell-free medium can be discharged through the outlet port. In some embodiments, the thickness of the pathology slide assembly can be compatible with a conventional optical microscope. For example, the pathology slide assembly can be optically transparent. For example, the pathology slide assembly can be sized to be mounted on a stage of an optical microscope for viewing. In some embodiments, the thickness of the pathology slide assembly is 1000 μm, 1100 μm, 1200 μm, 1300 μm, 1400 μm, or 1500 μm, or a range of thicknesses between any two of these values. In some embodiments, the pathology slide has a thickness of 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1100 μm, 1200 μm, 1300 μm, or 1400 μm, or a thickness within a range of any two of these values. In some embodiments, the pathology cover slip has a thickness of 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, or 250 μm, or a thickness within a range of any two of these values. In some embodiments, the surface of the pathology slide that defines the internal void comprises a cell-adhesive material. In some embodiments, the cell-adhesive material may be an adhesive gel, a protein coating, a sugar coating, or a nanomaterial. In some embodiments, cells delivered to the interior void can settle and adhere to the cell adhesive material. In some embodiments, the pathology slide and / or pathology coverslip may comprise an optically clear or crystal clear material.In some embodiments, the pathology slide and / or pathology coverslip may comprise plastic, glass, quartz, or ceramic, and in some embodiments, removal of the sealed coverslip may not be necessary for processing and effective visualization of the pathology slide assembly.

[0176] In some embodiments, the inlet and / or outlet ports may be configured to protrude from the pathology coverslip for easy access and easy connection. For example, the inlet and / or outlet ports may protrude from the pathology coverslip so that fittings (e.g., rubber or silicone tubing) can be attached to the inlet and / or outlet ports. In some embodiments, the protruding height of the inlet and / or outlet ports may be 0.4 cm, 0.5 cm, 0.6 cm, 0.7 cm, or 0.8 cm, or a protruding height within a range of any two of these values, or any other suitable height that allows easy access and connection.

[0177] In some embodiments, the internal cavity contains a single reservoir of uniform shape (a "pond" type reservoir). In such embodiments, there may be no microchannels within the internal cavity of the pathology slide assembly. In some embodiments, removal of the sealing coverslip may not be necessary for processing and effective visualization of the pathology slide.

[0178] In some embodiments, the pathology coverslip includes one or more microchannels, and the internal void includes a concave space defined by the one or more microchannels. In some embodiments, the one or more microchannels allow cells to pass through the interior (e.g., the dimensions of the one or more microchannels may be larger than the size of a cell). In some embodiments, the width of the concave space defined by each microchannel (e.g., the distance of the concave space between two adjacent microchannels of the pathology coverslip) is 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1100 μm, or 1200 μm, or a width within a range bounded by any two of these values. In some embodiments, the height of the recessed space defined by each microchannel (e.g., the distance from the surface of the pathology slide to the furthest surface of each microchannel) is 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, or 120 μm, or a range of any two of these values. In some embodiments, the sealed coverslip containing the microchannels may not need to be removed for processing and effective visualization of the pathology slide assembly. This provides the advantage of minimizing disturbance of stained adherent cells on the slide and reducing unintended loss of cellular specimens. Such an advantage may be advantageous in some embodiments, particularly when attempting to analyze rare target cells such as circulating tumor cells, of which only about 5-10 may be identified per slide.

[0179] In some embodiments, the surface of the pathology slide that defines the internal cavity may be nano-roughened. In some embodiments, the nano-roughened surface improves the capture of cells (e.g., cancer cells) in suspension. The use of nano-roughened surfaces for the capture of cancer cells that exhibit atypical morphology is described in Chen et al., Nanoroughened Surfaces for Efficient Capture of Circulating Tumor Cells without Using Capture Antibodies, ACS Nano. 7(1):566-75 (2013), and Chen et al., Nanoroughened adhesion-based capture of circulating tumor cells with heterogeneous expression and metastatic characteristics, BMC Cancer. 16:614, 1-12 (2016), which are expressly incorporated herein by reference in their entirety.

[0180] FIG. 2A shows a non-limiting embodiment of a pathology slide used in a pathology slide assembly. The pathology slide 200 may include an optically transparent and / or clear material configured for light microscopy, particularly for cytology purposes, which may be glass, plastic, quartz, ceramic, etc. In some embodiments, the pathology slide 200 may have a thickness that is compatible with a conventional light microscope. In some embodiments, the pathology slide 200 has a thickness of 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1100 μm, 1200 μm, 1300 μm, or 1400 μm, or a range of thicknesses between any two of these values. The pathology slide 200 may have an area 201 where specimen information may be handwritten or printed, as is also found on typical pathology slides. In some embodiments, the pathology slide 200 may have a cell adhesive material 202 to aid in the adhesion of cells introduced to the pathology slide 200 of the pathology slide assembly and encapsulate the cells. In some embodiments, the cell adhesive material may be an adhesive gel, a protein coating, a sugar coating, or a nanomaterial, including any cell adhesive material known in the art.

[0181] FIG. 2B shows two non-limiting embodiments of a pathology coverslip used in a pathology slide assembly. The pathology coverslip 203 may include an optically transparent and / or clear material configured for light microscopy, particularly for cytology purposes, which may be glass, plastic, quartz, ceramic, etc. In some embodiments, the pathology coverslip 203 has a thickness that is compatible with conventional light microscopes. In some embodiments, the pathology coverslip 203 has a thickness of 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, or 250 μm, or a range of thicknesses between any two of these values. In some embodiments, the pathology coverslip includes inlet and outlet ports for external access to the continuous internal volume (e.g., microchannels and reservoirs) of the pathology slide assembly. As shown in FIG. 2B, the inlet port may be used as an outlet port, and the outlet port may be used as an inlet port. In some embodiments, when liquid is introduced through the inlet port, the outlet port equalizes pressure with the external atmosphere to generate flow through the internal cavity of the pathology slide assembly. In some embodiments, the inlet and / or outlet ports 204 may protrude from the pathology coverslip for easy access and easy connection. For example, the inlet and / or outlet ports 204 may protrude from the pathology coverslip so that fittings (e.g., rubber or silicone tubing, etc.) can be attached to the inlet and / or outlet ports 204. In some embodiments, the protruding height of the inlet and / or outlet ports 204 may be 0.4 cm, 0.5 cm, 0.6 cm, 0.7 cm, or 0.8 cm, or a range of protruding heights between any two of these values, or any other suitable height that allows for easy access and connection.

[0182] In some embodiments, the pathology coverslip includes a single recessed region 205 that is less thick than the rest of the pathology coverslip, and this recessed region defines a single "pond" type reservoir of uniform shape within which the encapsulated cells are contained when sealed with the pathology slide as a pathology slide assembly.

[0183] In some embodiments, the pathology coverslip includes one or more microchannels 206, and the interior cavity of the pathology slide assembly including the pathology slide and the pathology coverslip includes a concave space defined by the one or more microchannels. In some embodiments, the one or more microchannels 206 allow cells to pass therethrough (e.g., the dimensions of the one or more microchannels may be larger than the size of a cell). In some embodiments, the width of the concave space defined by each microchannel 206 (e.g., the distance of the concave space between two adjacent microchannels of the pathology coverslip) is 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1100 μm, or 1200 μm, or a width within a range of any two of these values. In some embodiments, the height of the recessed space defined by each microchannel 206 (e.g., the distance from the surface of the pathology slide to the furthest surface of each microchannel) is 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm or 120 μm, or a height within a range bounded by any two of these values.

[0184] FIG. 2C shows two non-limiting embodiments of a pathology slide assembly. Pathology slide assembly 207 may use 1) a pathology cover slip with a single "pond" type reservoir or 2) a pathology cover slip with microfluidic channels. To construct the entire pathology slide assembly, the pathology slide and pathology cover slip may be stacked together and sealed, preferably with a waterproof permanent adhesive to prevent leakage of the contents of the pathology slide assembly (including cells, for example) during processing and diagnostic evaluation. When the pathology slide and pathology cover slip are stacked together and sealed, the interior cavity of the pathology slide assembly is accessible via inlet and outlet ports 204. However, once the desired cells are encapsulated in the prepared slide, the inlet and outlet ports 204 may be sealed for long-term storage purposes. Typically, the pathology slide assembly is sealed by filling the interior cavity with a compatible mounting medium to provide a uniform light path for visualization. Conventional mounting mediums also typically contain reagents that preserve cell morphology for storage.

[0185] FIG. 2D shows non-limiting cross-sectional and enlarged cross-sectional views of an exemplary pathology slide assembly. The pathology slide assembly 207 may include a pathology slide 200 and a pathology cover slip 203, configured similarly to a typical slide assembly. Enlarged view 208 (showing the entire pathology slide assembly, not a portion thereof) shows a cross-section including the pathology slide 200, the pathology cover slip 203, and a cell adhesive material 202 that may be disposed on the pathology slide 200. An internal void 209 may be defined by the inner surfaces of the pathology slide 200 and the pathology cover slip 203 of the pathology slide assembly 207. In some embodiments, this internal void may include a single "pond" type reservoir or may include multiple spaces defined by one or more microchannels 206, depending on the structure of the pathology cover slip 203. Cells 210 are shown adhering to the cell adhesive material 202. In preparing the final pathology slide assembly, the cells may be included in a mounting medium that enhances visibility for cytology purposes, such as mounting media known in the art. In some embodiments, the mounting medium may include, but is not limited to, α-pinene, toluene, and / or 2,6-butylated hydroxytoluene. In some embodiments, the mounting medium includes 25-60% w / w α-pinene, 40-72% w / w toluene, and 1-3% 2,6-butylated hydroxytoluene. In some embodiments, the mounting medium includes 27.5% α-pinene, 71.5% toluene, and / or 1% 2,6-butylated hydroxytoluene.

[0186] The pathology slide assemblies disclosed herein may be configured to be interconnected by connecting the outlet port of a first pathology slide assembly to the inlet port of a second pathology slide assembly. By flowing a cell suspension specimen through multiple pathology slide assemblies, the percentage of cells captured by the pathology slide assembly (the cell adhesive material 202) for visualization can be increased. By using multiple pathology slide assemblies, the area available for cell adhesion can be increased, which may be advantageous when the number of cells contained in the cell suspension specimen exceeds the number of cells that can be adequately adhered to a single pathology slide assembly. In some embodiments, the inlet port of the pathology slide assembly can be operably connected to the outlet port of the second pathology slide assembly via a connecting flow channel; and / or the outlet port of the pathology slide assembly can be operably connected to the inlet port of a third pathology slide assembly via another connecting flow channel. In some embodiments, the connecting flow channel can include a conventional material (e.g., rubber, silicone, etc.) and can flow liquids such as, for example, a specimen containing stained cells or a mounting medium through its internal flow channel. In some embodiments, both ends of the connecting channel can be attached to an inlet port and an outlet port, for example, by fitting to protruding inlet and outlet ports, respectively. In effect, liquid flows through the internal cavity of the first pathology slide assembly (where the first cell encapsulation occurs), then out the outlet port, through the connecting channel, and into the inlet port of the second pathology slide assembly to reach the internal cavity of the second pathology slide assembly (where the second cell encapsulation occurs).

[0187] Further disclosed herein are embodiments including a plurality of pathology slide assemblies including two or more of any of the pathology slide assemblies disclosed herein. In some embodiments, the two or more pathology slide assemblies can be serially connected via inlet and outlet ports of each pathology slide assembly, thereby fluidly connecting the internal cavities of the two or more pathology slide assemblies to form a continuous space. In some embodiments, an exposed inlet port of one of the two or more pathology slide assemblies and an exposed outlet port of another of the two or more pathology slide assemblies are not connected to another port, allowing access to the continuous space defined by the interconnected internal cavities.

[0188] 3A-B show non-limiting embodiments of a multiple pathology slide assembly. Pathology slide assembly 207 may represent any of the pathology slide assemblies disclosed herein. As described herein, the multiple pathology slide assemblies each have an inlet port and an outlet port, and may be connected or coupled to each other by connecting the outlet port of one pathology slide assembly to the inlet port of a second pathology slide assembly via a connecting channel 211. In some embodiments, the connecting channel 211 may be constructed of a conventional material (e.g., rubber, silicone, etc.) through which a liquid, such as a specimen containing stained cells or a mounting medium, can flow. In some embodiments, the inlet port and the outlet port may be constructed of the same material, and therefore either one of these two openings (the inlet port or the outlet port) may be coupled to either one of the openings of the second pathology slide assembly. Thus, the method of coupling the pathology slide assemblies is not limited to the specific configuration shown in FIG. 3A-B. As shown in the non-limiting embodiment of Figure 3B, each pathology slide assembly constituting the multiple pathology slide assemblies need not be the same, and may be composed of different types of pathology slide assembly structures as desired (e.g., structures having a reservoir-type internal void or a microchannel-type internal void, structures having microchannels of different widths, structures having different types of cell adhesive materials, etc.). Furthermore, although the non-limiting embodiment of Figures 3A-B shows three pathology slide assemblies connected together, any number of pathology slide assemblies may be connected together (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.).

[0189] As shown in FIGS. 4A-B, some non-limiting embodiments include a combination of staining devices. In some embodiments, the combination of staining devices includes any one of the devices disclosed herein and one or more pathology slide assemblies disclosed herein (multiple pathology slide assemblies may be coupled to each other as provided herein). In some embodiments, the combination of staining devices may include any one of the devices disclosed herein and any one of the pathology slide assemblies disclosed herein, where the specimen collection channel of the device can be operably connected to an inlet port of the pathology slide assembly. In some embodiments, the combination of staining devices may include any one of the devices disclosed herein and any one of the multiple pathology slide assemblies disclosed herein, where the specimen collection channel of the device can be operably connected to an exposed inlet port of the multiple pathology slide assemblies. In some embodiments, the specimen collection channel can be configured to fit into the inlet port, e.g., can be configured to be attached to a protrusion of the inlet port. In some embodiments, the specimen collection channel can be operably connected to the inlet port via another intermediate component (e.g., a tube, etc.). The device may be in a "vertical" configuration, as shown in the non-limiting embodiment of Figure 4A, or in a "horizontal" configuration, as shown in the non-limiting embodiment of Figure 4B.

[0190] Further disclosed herein is a method for encapsulating cells from a biological specimen obtained from a subject using a pathology slide assembly, or a plurality of pathology slide assemblies, as disclosed herein, the method comprising: a) flowing the suspension of cells into an interior cavity of a pathology slide assembly through an inlet port of the pathology slide assembly; or b) flowing the cell suspension into the contiguous space of the multiple pathology slide assembly through an exposed inlet port of the multiple pathology slide assembly; precipitating and / or adhering said cells to a surface of an internal cavity defined by a pathology slide or a plurality of pathology slides; Includes. In embodiments where the pathology slide may be coated with a cell-adhesive material, the cells may adhere to the cell-adhesive material. In some embodiments, the method further comprises flowing a washing reagent and / or a mounting medium into the internal space through an inlet port of the pathology slide assembly (or flowing a washing reagent and / or a mounting medium into the continuous space of the multiple pathology slide assemblies through an exposed inlet port of the multiple pathology slide assemblies) to exchange liquid components in the suspension of cells (or other liquids that may be present in the internal space, such as a washing reagent used in a previous step). In some embodiments, the cells may be retained in the internal space. In some embodiments, the liquid components in the suspension (or other liquids that may remain in the internal space, such as a washing reagent used in a previous step) may be drained through an outlet port (or an exposed outlet port of the multiple pathology slide assemblies). In some embodiments, the method comprises a final flowing step of flowing a mounting medium to fill the internal space (including the attached cells) with the mounting medium and provide a uniform light path for visualization. In some embodiments, after the final entry step, the inlet and outlet ports may be sealed (e.g., with adhesive, epoxy, glue, etc.) to maintain the cells in the mounting medium for storage. In some embodiments, the mounting medium may include a reagent that maintains cell morphology. In some embodiments, the method may further include visually inspecting the cells, which may have precipitated and / or adhered to the surface of the pathology slide or slides, by optical microscopy for the detection of atypical cells in the biological specimen and for the purpose of pathological diagnosis of disease. Typically, as described in some embodiments, to image the cells, a compatible mounting medium is filled into the internal void to ensure a uniform light path. In some embodiments, the cells may be fixed and / or permeabilized. In some embodiments, the cells may be stained with a cytomorphological stain, an immunocytochemical stain, or a chromogenic in situ hybridization stain, or any combination thereof.In some embodiments, the cells may be stained using a cytopathological multiple staining device disclosed herein and / or a method using the device. In some embodiments, the cytomorphological staining includes Diff-Quick staining, Papanicolaou staining, Wright-Giemsa staining, hematoxylin / eosin staining, or derivatives or variations thereof. In some embodiments, the cells include atypical cells. In some embodiments, the cells include cancer cells. In some embodiments, the cells include urinary sediment cells. In some embodiments, the urinary sediment cells may be stained for one or more bladder cancer specific biomarkers, which may be selected from the group consisting of S100P, p63, M344, LDQ10, 19A211, GATA-3, Ki-67, p16, Her-2, PD-L1, CTLA4, CK-17, CK-20, nmp-22, bladder tumor antigen (BTA), hTERT, and minichromosome maintenance protein 5 (MCM5).

[0191] In any of the embodiments provided herein, the patient may be a mammal. In some embodiments, the patient may be a human.

[0192] Separate Slide Chamber Assembly 5A-8 show another non-limiting embodiment of another staining chamber used in the device for performing cytopathological multiple staining. As shown in FIG. 5A and FIG. 5B, this another staining chamber 300 may include an upper part 301 and a lower part 302. In some embodiments, the upper part 301 can be detached from the lower part. In some embodiments, the lower part 302 may further include a porous membrane 303, which may be circular or elliptical in shape, and may have a size of up to 20 mm×20 mm in the case of a circular shape, and may have a size of up to 20 mm×40 mm in the case of an elliptical shape. In some embodiments, the upper part 301 and the lower part may be in contact with each other at a connecting part 304. As shown in FIG. 5A, the connecting part 304 may be sealed by a plastic molding part 305 disposed on the lower part 302, and this plastic molding part engages with the upper part 301. Also, as shown in FIG. 5B, the connecting part 304 may be sealed by a seal part 306 disposed on the lower part 302, and this seal part engages with the upper part 301. In some embodiments, the seal 306 may include an elastomeric material (e.g., rubber). In some embodiments, the upper portion of the dye chamber may include a protruding rim 307 at the top opposite the junction between the upper and lower portions.

[0193] 6A and 6B show a non-limiting example cross-section of another dyeing chamber 300 disassembled at the connecting portion 304 to separate the upper portion 301 from the lower portion 302. In some embodiments, the porous membrane 303 may remain attached to the lower portion when the upper and lower portions are separated. As shown in FIG. 6A, in some embodiments, the plastic molding 305 including the seal at the connecting portion 304 may remain attached to the lower portion 302 when the upper and lower portions are separated. Also, as shown in FIG. 6B, in some embodiments, the seal portion 306 including the seal at the connecting portion 304 may remain attached to the lower portion 302 when the upper and lower portions are separated. FIGS. 7A and 7B show the cross-section of FIGS. 6A and 6B with the connecting portion 304 and the seal portions 305, 306 clearly shown. FIG. 8 shows a close-up view of the cross-section of the connecting portion 304 and the seal portion 306 when the upper portion 301 and the lower portion 302 are assembled as shown in FIG. 5B. In some embodiments, the protruding rim 307 on the top of the staining chamber is configured as a fixture to pull out the cylindrical wall of the staining chamber after the staining process is completed, and the pulled out cylindrical wall is discarded (FIGS. 5A-B, 6A-B). In some embodiments, the stained cells may be exposed on the porous membrane 303 after the top 301 is removed. In some embodiments, after the top 301 is removed, the stained cells on the porous membrane 303 may be directly contacted with a pathology slide to transfer the stained cells onto the pathology slide. In some embodiments, the cell transfer process may be facilitated by blowing positive air pressure from below the porous membrane.

[0194] In some embodiments, the staining chamber 300 shown in Figures 5A-8 can be used as an alternative to the staining chamber 103 and semi-permeable membrane 104 shown in Figures 1D-E. In some embodiments, liquid reagents can be passed / aspirated downward into the lower portion 302 and discarded. In some embodiments, new solutions can be added from the top of the upper portion 301. In some embodiments, the staining chamber walls 301 can be removed after the staining process is completed.

[0195] Combined dyeing equipment 9-15 show non-limiting examples of a combination staining apparatus. In some embodiments, the combination staining apparatus may include a specimen inlet 400, an appearance inspection system 401, an operation status indicator 402, a control panel 403, a slide glass inlet 404, and a mouse and keyboard 405 (FIG. 9). The specimen inlet 400 may include a door for passing a specimen and placing it in the combination staining apparatus. The appearance inspection system 401 may include a hardware processor and / or display capable of image processing. The operation status indicator 402 may be capable of indicating the status of the combination staining apparatus. In some embodiments, the operation status indicator 402 may include one or more light sources capable of changing color to reflect the operation status of the combination staining apparatus. For example, the operation status indicator 402 may present a first color when the apparatus is ready to accept a specimen, may present a second color during processing of the specimen, and may present a third color when processing of the specimen is completed. In some embodiments, the operation status indicator 402 may flash, light, and / or turn off to indicate the operation status of the combination staining apparatus. The operating status indicator 402 may include a speaker that may sound a chime and / or an alarm to indicate the operating status of the combination stainer. In some embodiments, the control panel 403 may allow a user to start, pause, stop, and / or terminate the specimen processing. In some embodiments, the control panel 403 may be a touch screen. In some embodiments, the control panel 403 may display graphics indicating the progress of the specimen processing method being performed by the combination stainer. In some embodiments, the slide input slot 404 may allow a user to load one or more slides into the combination stainer. In some embodiments, the visual inspection system 401 may be controlled by a mouse and keyboard 405. In some embodiments, the control panel 403 may be controlled by a mouse and keyboard 405.

[0196] Referring to FIG. 10, in some embodiments, the combination staining apparatus may further include a reagent dispensing module 406. The reagent dispensing module 406 may be capable of dispensing one or more reagents into the staining chamber including the porous membrane 300. The reagent dispensing module 406 may include a reagent supply device 105 and / or a reagent pump 113. The reagent dispensing module 406 may be controlled by a hardware processor. In some embodiments, the staining chamber including the porous membrane 300 may be loaded into the combination staining apparatus, for example, from the sample input port 400. In some embodiments, the staining chamber may be disposed on a shaker 407. The shaker 407 may shake the staining chamber 300 to move the reagents in the staining chamber 300. The shaker 407 may be controlled by a hardware processor. Referring to FIG. 11, in some embodiments, the combination staining apparatus may simultaneously load up to 12 staining chambers. In some embodiments, the shaker 407 is disposed on a series of transport rails that allow the shaker 407 and the staining chamber 300 to be transported within the combination staining apparatus. The movement of the shaker 407 and the staining chamber 300 on the transport rail in the combination staining apparatus may be controlled by a hardware processor. In some embodiments, the combination staining apparatus includes a visual inspection field 408. The visual inspection field 408 may include a camera capable of transmitting images to the visual inspection system 401. The camera in the visual inspection field 408 may be capable of imaging the staining chamber 300 and may be capable of transmitting images of the staining chamber 300 to the visual inspection system 401 to determine completion of the staining. Referring to FIG. 12, in some embodiments, the combination staining apparatus further includes a gripper 409 capable of removing a portion of the chamber wall. In some embodiments, after the staining process is completed, the gripper 409 may engage a protruding rim portion 307 of the upper portion 301 of the staining chamber to pull the cylindrical wall of the staining chamber from the lower portion 302 and discard the pulled upper portion 301.With reference to FIG. 13, in some embodiments, the transport arm 410 can transfer the stained cells to the slide by pressing the exposed stained cells against the slide containing the cell adhesive material. In some embodiments, the cell transfer process can be facilitated by pressing the cells on the porous membrane upwards using a pump capable of generating positive air pressure from below the porous membrane. With reference to FIG. 14, in some embodiments, the combination staining device further comprises a waste collection hole or waste collection reservoir 411. In some embodiments, the gripper 409 can discard the top of the staining chamber into the waste collection hole or waste collection reservoir 411. Reagents (e.g., reagents used in the staining process) may be removed from the staining chamber 300. With reference to FIG. 15, in some embodiments, the combination staining device further comprises a liquid waste collection device 412. The liquid waste collection device 412 can remove used reagents and / or liquid waste from the staining chamber 300. In some embodiments, the liquid waste collection device 412 comprises a pump capable of generating a relative negative pressure compared to the pressure at the top 301 of the staining chamber. In some embodiments, a liquid waste collector 412 can remove used reagents from the staining chamber 300 to a waste collection well or waste collection reservoir 411. In some embodiments, the process of producing stained pathology slides from liquid specimens is automated. In some embodiments, the process of producing stained pathology slides from liquid specimens is completed in a closed system. In some embodiments, specimen processing occurs in about 3 hours.

Claims

1. 1. An apparatus for performing multiple cytopathological staining, comprising: a) a staining container configured to contain an aqueous cell culture medium, a staining reagent, a washing reagent and / or a slide mounting medium, and cells from a biological specimen obtained from a patient, and configured to provide stained cells by alternately performing staining and washing steps; b) an extraction vessel configured to receive a used aqueous cell culture medium, a used staining reagent, and / or a used washing reagent from the staining vessel, and configured to allow the used aqueous cell culture medium, the used staining reagent, and / or the used washing reagent to be removed; Including, the staining container and the extraction container are operably separated via a semipermeable membrane, which is permeable to the aqueous cell medium, the staining reagent, the washing reagent and / or the mounting medium, but not to the cells; Device.

2. 10. The apparatus of claim 1, wherein the semi-permeable membrane comprises pores configured to allow liquids and small molecules to pass through the semi-permeable membrane but not the cells, and wherein the semi-permeable membrane is configured to retain the cells within the staining vessel.

3. 3. The device of claim 2, wherein the diameter of each pore is 0.05 μm, 0.1 μm, 0.2 μm, 0.22 μm, 0.3 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 3 μm, 5 μm, or 10 μm, or a range defined by any two of these values.

4. The device of any one of claims 1 to 3, wherein the staining reagent comprises a cytomorphological stain, an immunocytochemical stain, or a chromogenic in situ hybridization stain, or any combination thereof.

5. 5. The device of claim 4, wherein the cytomorphological stain comprises a Diff-Quick stain, a Papanicolaou stain, a Wright-Giemsa stain, a hematoxylin / eosin stain, or a derivative or modification thereof.

6. The apparatus according to any one of claims 1 to 3, wherein the semipermeable membrane is configured to move the staining reagent used in each staining step and the washing reagent used in each washing step in the staining container to the extraction container by passive diffusion, or to move the staining reagent and the washing reagent to the extraction container when the extraction container is under negative pressure relative to the staining container, and the negative pressure may be applied by an extraction pump.

7. The apparatus according to any one of claims 1 to 3, wherein the staining container and the extraction container are configured to be separated or blocked by a shutter that prevents or inhibits the aqueous cell culture medium, the staining reagent, and / or the washing reagent from moving through the semipermeable membrane between the staining container and the extraction container.

8. 4. The apparatus of claim 1, further comprising a reagent supply source configured to provide the aqueous cell culture medium, the staining reagent, the washing reagent, and / or the slide mountant to the staining container, wherein the reagent supply source may be operably connected to a reagent pump configured to move the aqueous cell culture medium, the staining reagent, the washing reagent, and / or the slide mountant from the reagent supply source to the staining container, and the aqueous cell culture medium may contain the cells.

9. 4. The device of claim 1, further comprising a specimen collection channel configured to collect the stained cells, e.g., for mounting purposes, wherein the stained cells may be contained in the slide mounting medium, and wherein the specimen collection channel is configured to collect the stained cells using negative pressure.

10. A method for multiplex cytopathological staining of cells derived from a biological specimen obtained from a patient, the method comprising staining the cells by one or more staining steps using an apparatus according to any one of claims 1 to 3.

11. 11. The method of claim 10, wherein the staining step comprises staining the cells with a cytomorphological stain, an immunocytochemical stain, or a chromogenic in situ hybridization stain, or any combination thereof.

12. 11. The method of claim 10, wherein the cytomorphological stain comprises Diff-Quick stain, Papanicolaou stain, Wright-Giemsa stain, hematoxylin / eosin stain, or a derivative or modification thereof.

13. 1. A pathology slide assembly comprising a pathology slide and a pathology coverslip, the pathology slide and the pathology coverslip are configured to be sealed together; the pathology slide and the pathology coverslip define an interior cavity accessible through an inlet port and an outlet port of the pathology slide assembly; the internal cavity is configured to receive a suspension of cells from a biological specimen obtained from a patient through an inlet port and to discharge a cell-free medium through an outlet port. Pathology slide assembly.

14. 14. The pathology slide assembly of claim 13, wherein the thickness of the pathology slide assembly is compatible with an optical microscope.

15. 15. The pathology slide assembly of claim 13 or 14, wherein the surface of the pathology slide defining the internal void comprises a cell-adhesive material, which may be an adhesive gel, a protein coating, a sugar coating, or a nanomaterial configured to immobilize the cells.

16. 15. The pathology slide assembly of claim 13 or 14, wherein the internal cavity comprises a single reservoir of uniform shape.

17. 15. The pathology slide assembly of claim 13 or 14, wherein the pathology coverslip is configured to include one or more microchannels, and the internal void comprises a concave space defined by the one or more microchannels, the one or more microchannels being configured to allow the cells to pass therethrough.

18. 15. The pathology slide assembly of claim 13 or 14, wherein a surface of the pathology slide or a surface of the coverslip, or both, preferably the surface of the pathology slide defining the internal void, is nano-roughened, and the nano-roughened surface improves capture of the cells in the suspension.

19. 15. The pathology slide assembly of claim 13 or 14, wherein the inlet port is configured to be operably connected to an outlet port of a second pathology slide assembly via a connecting channel, and / or the outlet port is configured to be operably connected to an inlet port of a third pathology slide assembly via another connecting channel.

20. A multiple pathology slide assembly comprising two or more pathology slide assemblies according to claim 13 or 14, the two or more pathology slide assemblies are connected in series via the inlet port and the outlet port of each pathology slide assembly, thereby fluidly communicating the internal cavities of the two or more pathology slide assemblies to form a continuous space; an exposed inlet port of one of the two or more pathology slide assemblies and an exposed outlet port of another of the two or more pathology slide assemblies are not connected to another port, allowing access to the continuous space; Multiple pathology slide assembly.

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