WO2022042777A1 - Procédé de préparation d'une puce de pathologie de cellules tumorales circulantes dans le sang - Google Patents
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Definitions
- the invention relates to the detection of circulating tumor cells (Circulating Tumor Cell, CTC) in the blood, and more particularly to the staining of circulating tumor cells in a blood sample, and then making them in a pathological chip for morphological diagnosis and biological identification.
- CTC circulating Tumor Cell
- the present invention also relates to a pathology chip and a kit for performing the above method.
- cytopathology (cytology) pathology slides are generally prepared by using patient blood and other body fluids (1) to smear directly on the slide, (2) or to fix the cells in the liquid on the slide by centrifugal force ( cytospin), (3) or through the centrifugation process, the cells in the liquid are formed into a precipitate (cell block), and then subjected to routine pathological section processing, and then the pathologist observes the cells under a microscope for cytopathological diagnosis.
- Circulating tumor cells exist in the blood of cancer patients at a very low frequency (on average 1 CTC/ml blood). If the smear preparation method is used to detect 10 ml of blood of cancer patients, 200 blood smears need to be prepared, which cannot be promoted clinically. The same problem exists with centrifugation to prepare cytospins. If all cell blocks are used to prepare pathological sections, only a small number of precipitated cells can be observed on the section, which is not suitable for the detection of very small amount of circulating tumor cells. Therefore, the conventional cytopathological slide preparation method obviously cannot screen the circulating tumor cells in the blood of patients in clinic, and thus cannot detect the tumor or tumor metastasis.
- CTCs Circulating tumor cells
- CN109507427A discloses a method for enriching CTCs, wherein by adding biotin-conjugated CD45 monoclonal antibody and streptavidin-conjugated magnetic beads, leukocytes are adsorbed on magnetic beads and then enriched by removing magnetic beads circulating tumor cells.
- magnetic beads and magnetic force will destroy tumor cells, and the process of sedimentation of leukocytes will entrain rare tumor cells and cause tumor cells to be lost, making the originally rare CTC cells less.
- the magnetic beads will make physical contact with CTCs during the enrichment process, and the physical involvement of CTCs through the bound membrane proteins in the magnetic field will affect the morphological characteristics of CTCs and seriously affect the pathomorphological diagnosis of cells.
- cytopathological identification of these rare tumor cells is required.
- Conventional staining methods for cytopathology include Papanicolaou, Wright-Giemsa, H&E, and the like.
- Diff-Quik is prepared by the rapid staining method recommended by the World Health Organization (WHO). Similar to Wright-Giemsa, it is improved by the principle of Pasteur technology. of.
- the staining results can show the characteristics of the cell membrane, such as cell folds, cell vesicles; cytoplasmic mucus, fat granules, neuroendocrine granules; the characteristics of the nucleus, including the shape and size of the nucleus, the number and shape of the nucleoli, the number of chromosomes, staining
- the texture of the texture provides cytopathologists with cytomorphological diagnostic information.
- the dyes of conventional cytopathological staining methods will affect the binding of the first antibody or the first probe to the relevant target, so these stainings need to be washed in order to better realize the immunohistochemical staining of biomarkers.
- higher concentrations of protein antibodies or nucleic acid probes are often required for staining, which can easily cause false positives and mislead clinical diagnosis, treatment and prognosis.
- the cell smear is in an open environment during the staining process. During the staining process, the staining liquid should be continuously replaced, rinsed, and then discarded.
- CN109239030A discloses a method of immunofluorescence staining of enriched CTC cells to detect tumor markers.
- cells are first fixed on a slide, and the slide is washed several times in an open system before staining, so that cells will be lost during the staining process, making the originally rare CTCs cells become fewer.
- the patent application uses immunofluorescence staining, which requires fluorescence microscopes and other special scanning equipment, and cannot provide morphological characteristics of cells that can be used for pathological diagnosis.
- the invention presents the circulating tumor cells in the peripheral venous blood of cancer patients with complete morphology on the pathological chip, so that the same target cell can be displayed under a common optical microscope, and the morphological characteristics of the cells that can be used for pathological diagnosis can be provided at the same time. And tumor markers, showing the number of circulating tumor cells, pathological characteristics, and tumor-specific antigen expression, to guide the clinical diagnosis and treatment of cancer.
- the present invention is realized by three parts: sample pretreatment, cell immunohistochemical staining and/or chromogenic in situ hybridization, and pathological chip fabrication.
- the core of the sample pretreatment process of the present invention is to greatly reduce the number of red blood cells and white blood cells in the blood on the premise that the morphology and quantity of circulating tumor cells are not affected, so that the targeted circulating tumor cells can be prepared in one or more On the pathology chip, cytopathological identification can be carried out directly under the ordinary light microscope, which is suitable for the routine work flow of pathology department.
- the invention relates to an innovative cell multiple staining method in the field of cytopathology, which can simultaneously display detailed morphological characteristics of cells that can be used for pathological diagnosis on the same sample cell, and the expression of one or more specific biomarkers. express information.
- the staining results are suitable for observation under a common light microscope, and the expression of one or more specific biomarkers on the same cell can be simultaneously observed under the premise of meeting the pathologist's requirements for cytopathological diagnosis.
- the staining process does not lose cells and is suitable for multiple staining of a small number of cell samples.
- the invention solves the technical difficulties of routine cytopathological staining and multiple staining of biological markers, and after performing immunohistochemistry or nucleic acid chromogenic in situ hybridization staining on cells, routine cytopathological staining is performed on the same cell. Finally, on the same cell, under the ordinary light microscope, the morphological characteristics of the cells at the diagnostic level and the multiple information of the biomarkers can be obtained at the same time.
- Staining results can show morphological features required for cytopathological diagnosis, including but not limited to: cell membrane morphology such as membrane folds and cell morphology; cytoplasmic mucus, fat granules, neuroendocrine granules; nuclear features, including nuclear Morphology and size, number and shape of nucleoli, texture of chromatin.
- the method clearly provides cytopathologists with basic information of cytomorphological diagnosis, and at the same time combines the expression information of one or more biomarkers on the same cell, which helps to improve the accuracy of cytopathological diagnosis.
- cytopathological sample is a cell, and each cell is individually adsorbed or placed on a pathological glass slide by smear and other methods to form a so-called cell smear.
- Cell smear samples are kept in an open environment during the staining process, during which the staining solution is constantly replaced, rinsed, and then discarded. With the continuous replacement and disposal of the staining solution and washing solution, the cell samples on the pathological slides will inevitably fall off and be lost, reducing the sensitivity and accuracy of the staining results, and prone to false negatives, especially when the sample contains a small number of cells. case.
- Routine cytopathological immunohistochemical staining and nucleic acid chromogenic in situ hybridization take the pathological glass slide as the carrier of the cell sample, which requires the cell sample and the pathological glass slide to be in direct and close contact, and there should be no gap between the cells to cause the cells to be removed from the glass. The sheet falls off.
- the staining solution cannot reach the contact position between the cells and the glass slide, and a blind spot for staining is formed at the contact position between the cells and the glass slide, so that the cells cannot be fully stained in all directions, leaving dead spots for staining, which affects the sensitivity and accuracy of the staining results. .
- the chromogenic reaction of immunohistochemistry and chromogenic in situ hybridization is carried out on the pathological glass slide, and the optically visible chromogenic product is produced by the catalytic reaction, which is concentrated near the chromogenic enzyme at the cellular level and becomes a targeted biomarker expression signal.
- the precipitated chromogenic product is opaque to light. If too much and densely deposited on the cells, the chromogenic product will cover the characteristics of cell phase morphology when observed under an ordinary optical microscope, affecting the pathological diagnosis of cells.
- the inventor adopts a new and improved staining method of cell immunohistochemistry and nucleic acid chromogenic in situ hybridization.
- the pathological glass slide is not used as a carrier for staining, but the sample cells are placed in a container and stained in the form of a cell suspension. After the cells are stained, the smear is fixed and observed under a microscope. The order in which the sample cells are fixed is the opposite of the conventional method.
- the inventors place the cells in a container, and complete the incubation of the primary antibody or nucleic acid probe in the cell suspension, and the staining processes such as color reaction.
- the cell samples are pelleted by centrifugation, or the cell samples are passed through the microfluidic channel of protein adsorption, or the cell samples are contained in the micropores that can only pass high-molecular proteins but not intact cells.
- Membrane container and other means so that there are no targeted cells in the discarded supernatant wash, which greatly reduces the loss of clinical cell samples during the staining process.
- the stained cells are spread on the pathological glass slides to ensure the stability and reliability of the results.
- the inventor did not use the pathological glass slides as carriers during the staining and chemical development process, and always kept the cells in a suspension state, fully immersed and contacted in different conditions.
- the dyeing reagent there is no dyeing blind area and dead angle, which can improve the dyeing effect of cells, improve the sensitivity of dyeing, and reduce the false negative rate of dyeing, which is of obvious help.
- the improved chemical color developing method makes the color product evenly distributed.
- Conventional immunohistochemical staining and nucleic acid chromogenic in situ hybridization are carried out on pathological slides, and the chromogenic reaction is carried out on fixed tissues.
- the chromogenic precipitates produced by catalysis are easy to accumulate in local areas, because they are opaque and can be covered.
- the morphological characteristics of the fine structure of cells affect the morphological diagnosis of cytopathology.
- the inventor continuously shakes the cells suspended in the liquid, so that the chromogenic precipitated products produced by catalysis are evenly dispersed, and the unobstructed optical path is not affected by concentrated accumulation.
- the chromogenic products produced by catalysis are reasonably distributed on the cells, which can not only display the expression information of biomarkers, but also do not hinder the diagnosis of cell morphology, and greatly improve the accuracy of the pathologist's diagnosis of cell phase morphology.
- the invention can fully wash the single cells in the cell suspension without blind spots and dead corners in all directions, and at the same time, because there are no such bad links as non-specific deposition and edge effects on the pathological glass slides, it can greatly reduce the damage on the pathological glass slides.
- Nonspecific background staining noise It has obvious help to improve the staining effect of cells, improve the sensitivity of staining, and reduce the false positive rate of staining results.
- Tumor pathology chip This pathological chip system is independent and has the characteristics of one-time use, avoiding cross-contamination and improving the accuracy of detection. Chips can be connected in series with each other. One or more pathology chips can be fabricated according to the number of cells in the sample, which can ensure that all circulating tumor cells are presented on the pathology chip.
- CN109239030A and CN102313813A disclose the observation of tumor cell slides with a microscope, but the slides do not have the microfluidic channels and adsorption materials in the chip of the present invention, so further separation of CTCs and leukocytes and adsorption of leukocytes cannot be achieved.
- the refraction of the microfluidic channel is avoided and the optical path is kept unobstructed, which can be directly observed under a conventional microscope, and the multi-stained blood circulating tumor cells on ordinary pathological slides can be counted, which is in line with routine pathology workflow.
- the present invention can also use digitally scanned electronic pathology slides to perform the above method. After scanning the plain slides, the tumor cells on the electron pathology slides were counted.
- those skilled in the art can judge tumor recurrence, prognosis and malignancy; monitor the disease course of known tumor patients; determine the specific tumor Drug susceptibility, thereby guiding the choice of chemotherapy, targeted therapy, radiation therapy, and immunotherapy.
- the present invention relates to the following technical solutions:
- the present invention relates to a multiple staining method for immunohistochemical staining and/or nucleic acid chromogenic in situ hybridization staining and cytomorphological staining that can be used for pathological diagnosis on circulating tumor cells (CTCs) to be fabricated into pathological chips, including :
- c) Perform cytomorphological staining of the cells for pathological diagnosis in the container, and then spread the multiple-stained cells on the pathological slides; alternatively, stain the cells with immunohistochemical staining and/or nucleic acid chromogens Cells stained by site hybridization were plated on pathological slides for morphological staining that could be used for pathological diagnosis.
- steps ii), iv) and vi) remove unbound said first antibody and/or first nucleic acid probe, remove unbound said chromogenic enzyme-conjugated second Antibody and/or chromogenic enzyme-labeled second nucleic acid probe and removal of excess chromogenic substrate through the following steps:
- the morphological staining of cells that can be used for pathological diagnosis is selected from the group consisting of Diff-Quik staining, Papanicolaou, Wright-Giemsa staining, and H&E staining, preferably Diff -Quik staining.
- the present invention can simultaneously display detailed morphological characteristics of cells that can be used for pathological diagnosis and the expression information of one or more specific biomarkers on the same sample cell.
- the staining results are suitable for observation under a common light microscope, and the expression of one or more specific biomarkers on the same cell can be simultaneously observed under the premise of meeting the pathologist's requirements for cytopathological diagnosis.
- the staining process does not lose cells and is suitable for multiple staining of a small number of cell samples.
- the cell smear can be directly observed under a conventional microscope without other equipment.
- the pathological diagnosis characteristics of the nucleus, cytoplasm, and cell membrane can be observed under a conventional microscope, and the tumor-specific antigen expression site, including the nucleus, cytoplasm, and cell membrane, can be determined.
- the immunofluorescence method in the prior art can observe the outline of the extracellular shape, it cannot provide the morphological characteristics of cells that can be used for pathological diagnosis, and it cannot be used for pathological morphological diagnosis.
- the synergistic effect of these two diagnostic indicators is obvious.
- the expression of specific tumor markers can help the final tumor cell pathomorphological diagnosis and improve the sensitivity of the diagnosis.
- a suspicious positive signal or non-specific background noise signal appears in tumor marker staining, it can be combined with the diagnosis of cell morphology to exclude false positive signals on non-tumor cells and improve the accuracy and specificity of diagnosis.
- the original double staining technology of the invention greatly improves the sensitivity of detection, reduces false positives, and improves the specificity of detection. It is of great significance in clinical testing, especially in the field of tumor screening.
- the present invention relates to a chip for observing CTCs under a microscope, the chip comprising:
- the transparent filling liquid is filled in the microfluidic channel.
- the filling liquid avoids the lens effect caused by the microfluidic channel and ensures the optical path of the optical microscope Unobstructed, so that the number, morphological characteristics of CTCs, and staining results of immunohistochemistry and/or chromogenic in situ hybridization can be observed under a microscope.
- the size of the slide is a conventional size, eg, about 75 mm by 25 mm.
- an area (oval or rectangular) of about 40-50 x 20-25 millimeters in the slide contains cell-adsorbing material.
- the cell-adsorbing material comprises nanomaterials, such as a rough surface of 100-200 nanometers.
- the coverslip is about 40-50 X 20-25 mm in size and about 0.15-0.25 mm thick.
- the microfluidic channel is about 0.5-1.5 mm wide and about 50-100 microns high.
- the microfluidic channel can be a serpentine single channel, or a straight multi-channel.
- the protruding height of the inlet and outlet valve sites may be about 0.4-0.8 cm to facilitate the pumping of the cell suspension by the device.
- the positions of the liquid inlet and the liquid outlet do not block the movement trajectory of the conventional microscope objective lens when it is converted. Multiple chips can be connected in series with each other by connecting the liquid inlet and the liquid outlet.
- the present invention relates to a method for making a circulating tumor cell (CTC) pathological chip, comprising:
- this step uses two one or more of the above-mentioned chips connected in series with each other;
- the present invention relates to a method for making a circulating tumor cell (CTC) pathological chip, comprising:
- this step uses two one or more chips as described above connected in series with each other;
- step i) comprises
- step ii) comprises:
- Step ii) is carried out by the multiple staining method described in the first aspect.
- the filling composition comprises the following ingredients:
- ⁇ -pinene which is a class of natural organic compounds with the same skeleton structure, belongs to bicyclic monoterpenes, and has the molecular formula C 10 H 16 ;
- the weight percentages of the three components a), b) and c) can be respectively: 25%-60%, 40%-70% and 1-3%.
- the weight percentages of the three components a), b) and c) are 27.5%, 71.5% and 1.0%, respectively.
- the present invention also relates to a method for ensuring an unobstructed optical path of a chip during the fabrication of a pathological chip, comprising: filling the microfluidic channels in the chip with the composition described above, thereby avoiding lens effects.
- the chip system is independent and single-use to avoid cross-contamination of samples.
- This closed system does not need to be opened, and the adsorbed cells are not moved to avoid cell loss and simplify the operation process.
- Multiple chips can be connected in series with each other by connecting the liquid inlet and the liquid outlet.
- the chip size and thickness are suitable for direct observation by conventional optical microscopes, including 40X high magnification objective. It conforms to the routine work flow of the pathology department and does not require any special equipment to be added to the microscope.
- Microfluidic channels can be filled with special liquids that solidify and protect stained cells for a long time. Its refractive index is the same as that of the cover glass material, which ensures the smooth optical path of the microscope and avoids refraction affecting the observation.
- the adsorption material has a specific adsorption function to tumor cells.
- Immunohistochemically stained sections can be stored for a long time, which meets the routine requirement of 7 years of preservation of clinical medical evidence.
- immunofluorescence in the prior art can only be stored for one month, and then cannot be observed due to fluorescence decay, and cannot be stored for a long time as medical evidence.
- the present invention also relates to a method for counting circulating tumor cells, including using an optical microscope to count the circulating tumor cells in the circulating tumor cell pathology chip produced by the method of the present invention; or digitally counting the circulating tumor cell pathology chip produced by the method of the present invention Scanned and counted tumor cells on electron pathology slides.
- the present invention also relates to methods for tumor screening, diagnosis, curative effect judgment and prognosis, including the shape, size, number or tumor marker information of multiple stained tumor cells in the circulating tumor cell pathology chip prepared according to the method of the present invention to judge the tumor Likelihood of recurrence or malignancy, or to determine the drug sensitivity of a tumor to guide the choice of chemotherapy, targeted therapy, radiation therapy, or immunotherapy, or to perform tumor screening in the general population.
- Fig. 1 is the design drawing of the chip of the present invention.
- use regular size pathology slides 75mm X 25mm, 1mm thick.
- the 40 X 20 mm area (oval or rectangular) contains the cell-adsorbing material (nanomaterial) (above). It is covered with a 50X25 mm coverslip with a thickness of 150-200 ⁇ m.
- the microfluidic channel is designed with a width of 500-1500 microns and a height of 50-100 microns (middle image).
- the glass slide and the cover glass form a closed system, and the cell suspension passes through the microfluidic channel to ensure that the rare tumor cells are fully contacted with the adsorption material to be adsorbed on the glass slide.
- valve parts of the liquid inlet and the liquid outlet of the microfluidic channel are protruded or recessed, and the height can reach 0.5 cm, which is convenient for the equipment to pump the cell suspension and form a series connection.
- the positions of the liquid inlet and the liquid outlet do not block the movement trajectory of the conventional microscope objective lens when it is converted.
- the location can be on the sides, or on top of the coverslip (below).
- Figure 2 shows enrichment, double staining, and pathology chip coating of bladder cancer tumor cell line T24 in blood.
- Three tumor cells (marked by red arrows) in one field of the high-power microscope image were stained brown by immunohistochemistry with CK-7 monoclonal antibody and Diff-Quik double staining. Diff-Quik-stained remaining blood cells, including neutrophils, and lymphocytes are seen in the background.
- EDTA anticoagulant tube 10 ml blood collection tube to collect venous blood from the patient.
- a cell suspension containing 100 human urothelial carcinoma cell line T24 was added.
- the circulating tumor cell pellet obtained in Example 1 was diluted and suspended in 1 ml of PBS (pH 7.4) buffer, and placed in a 10 ml plastic test tube.
- Diff-Quik staining of the cell suspension Add 1 ml of Diff Rapid Stain No. 2 (source Fisher Scientific, product #22750012) diluted to 5% in PBS buffer, incubate for 20 seconds and then centrifuge at 1000 rpm for 5 minutes to remove excess chromogenic reagent. Add 1 ml of Diff Rapid Stain No. 1 (source Fisher Scientific, product #22750012) diluted to 5% in PBS buffer, incubate for 20 seconds and then centrifuge at 1000 rpm for 5 minutes to remove excess chromogenic reagent. A cell suspension was prepared by adding 200 microliters of PBS buffer.
- the chip is designed as follows:
- the 40X20mm area contains the cell adsorption material (100nm rough surface);
- microfluidic channel is a serpentine single channel
- the glass slide and the cover glass form a closed system, and the cell suspension passes through the microfluidic channel to ensure that the rare tumor cells are fully contacted with the adsorption material to be adsorbed on the glass slide;
- valve parts of the liquid inlet and the liquid outlet are prominent and the height is 0.5 cm, which is convenient for the equipment to pump the cell suspension;
- Example 2 The liquid containing the stained circulating tumor cells and part of the remaining leukocytes obtained in Example 2 was pumped into the microfluidic channel of the above-mentioned pathological chip through the liquid inlet on the above-mentioned glass slide;
- the liquid After the liquid passes through the microfluidic channel, it flows out through the liquid outlet, and the CTC is adsorbed on the adsorption material of the glass slide to form a monolayer cell adsorption layer.
- Transparent filling liquid fills the microfluidic channel in the microfluidic channel to ensure the smooth optical path of the optical microscope.
- the filling liquid contains the following ingredients:
- ⁇ -pinene which is a class of natural organic compounds with the same skeleton structure, belonging to bicyclic monoterpenes, molecular formula C10H16;
- the weight percentages of the three components a), b), and c) are 27.5%, 71.5%, and 1.0%, respectively.
- the prepared pathological sections were observed directly under a conventional microscope: the tumor cells were stained brown (see Figure 2).
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Abstract
La présente invention se rapporte à un procédé de coloration multiple pour une puce de pathologie de cellules tumorales circulantes dans le sang et un procédé de fabrication correspondant. En particulier, la présente invention se rapporte à la réalisation d'une coloration à base de liquide sur des cellules tumorales circulantes dans un échantillon de sang, et, ensuite, à la préparation des cellules dans une coupe de pathologie pour un test. La présente invention se rapporte également à une puce de pathologie utilisée pour exécuter le procédé décrit, et à un procédé utilisant ladite puce pour le criblage, le diagnostic, la détermination d'un effet curatif et le pronostic de tumeurs.
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