Immunofluorescence staining and Papanicolaou staining combined detection method, kit and system

Abstract

The present invention discloses a combined detection method, kit and system of immunofluorescence staining and Pap staining, and belongs to the technical field of diagnostic reagents. The method comprises the following steps: fixing exfoliated cells to obtain a first sample sheet; staining the first sample sheet by immunofluorescence staining reagent, and sealing the sheet by a cover glass to obtain a first stained sheet; obtaining the staining result of the first stained sheet; soaking the first stained sheet in 90-100°C hot water for 3-10 minutes, removing the cover glass to obtain a second sample sheet; staining the second sample sheet by Pap staining solution, and sealing the sheet by neutral gum to obtain a second stained sheet; obtaining the staining result of the second stained sheet. Performing two kinds of staining on the same sample sheet in sequence, namely, combined detection of immunofluorescence staining and Pap staining, better improves the accuracy of cytological diagnosis results and can improve the authenticity of the results; and the two stainings will not interfere with each other.

Description

Technical Field

[0001] The present invention relates to the technical field of diagnostic reagents, and in particular to a combined detection method, a kit and a system of immunofluorescence staining and Papanicolaou staining. Background Art

[0002] The detection or diagnosis of exfoliated cells is a conventional method for disease diagnosis, such as immunofluorescence staining and Papanicolaou staining. For example, early screening and diagnosis of various cancers have always been the focus of the medical field and the key to improving the treatment rate and survival rate of patients. At present, early screening cell diagnosis for cancer mainly relies on traditional exfoliated cell staining, that is, diagnosis after Papanicolaou staining, but it requires high diagnostic experience of doctors.

[0003] Papanicolaou staining is one of the best staining methods for exfoliated cells, and it is suitable for specimens of epithelial cells and mesothelial tissues. Papanicolaou staining cannot clearly display the nuclear structure, and has the characteristics of obvious color separation, good transparency, and bright cytoplasmic color. However, it is difficult to make judgments for specimens with a small number of cells, such as urine, especially hematuria specimens, which are covered by a large number of hemoglobin cells and exfoliated cells, which further affects the interpretation results; for thyroid fluid-based specimens, specimens with a small number of cells and many clusters require a high level of manual reading. A large number of experiments have shown that Papanicolaou staining is not very sensitive to positive cells, and the intensity of doctor reading is high. Different doctors may make different diagnostic results for the same slide.

[0004] Immunofluorescence staining mainly stains nucleic acids in the cell nucleus, and uses a fluorescence microscope in the B band (wavelength 470-495nm) to observe that DNA and RNA show different fluorescence to the dye. When cells proliferate and differentiate actively or show polyploidy, the entire cell shows bright fluorescence under a fluorescence microscope due to the accumulation of a large amount of nucleic acids in the cell nucleus. Cells that are not actively proliferating and differentiating will appear dim. Immunofluorescence staining greatly reduces the intensity of film reading.

[0005] The interpretation of the results of the above-mentioned single detection method of exfoliated cells depends on personal experience, and the detection results of various methods may be inconsistent, resulting in low accuracy and reliability of the detection results. Summary of the invention

[0006] In view of the above technical problems existing in the prior art, the present invention provides a combined detection method, kit and system of immunofluorescence staining and Papanicolaou staining, which detect the same sample sheet by combining the two staining methods.

[0007] The first aspect of the present invention provides a combined detection method of immunofluorescence staining and Papanicolaou staining, comprising the following steps: fixing exfoliated cells to obtain a first sample sheet; staining the first sample sheet with an immunofluorescence staining reagent and sealing the sheet with a cover glass to obtain a first stained sheet; obtaining the staining result of the first stained sheet; soaking the first stained sheet in 90-100°C hot water for 3-10 minutes, removing the cover glass to obtain a second sample sheet; staining the second sample sheet with a Papanicolaou staining solution, sealing the sheet with a neutral gum to obtain a second stained sheet; obtaining the staining result of the second stained sheet.

[0008] Preferably, the method for identifying staining results: marking the cell types of the staining results and constructing a staining result set; training the staining result set based on a machine learning method to obtain a cell classification model; and analyzing the staining results based on the cell classification model to obtain the types of cells in the staining results.

[0009] Preferably, the cell types include diploid cells, neutrophils, inflammatory cells, lymphocytes, microorganisms, inflammatory cells, squamous cells, squamous cell carcinoma gland cells and adenocarcinoma cells.

[0010] Preferably, the immunofluorescence staining reagent includes liquid A, liquid B and liquid C, and the method for staining the first sample piece includes:

[0011] Add solution A dropwise onto the visible components of the first sample slide, shake gently to cover the entire smear area, and stain for 1-3 minutes;

[0012] Pour off excess dye, add solution B, shake gently to cover the entire smear area, and separate the colors for 30-60 seconds;

[0013] Remove excess residual liquid by suction, add liquid C to seal the slide, and obtain the first stained slide.

[0014] Preferably, the method of staining the second sample piece with a Papanicolaou staining solution comprises:

[0015] Soak in EA50 staining solution for 10 seconds;

[0016] Decolorize with 9.95% ethanol, 95% ethanol, anhydrous ethanol, and anhydrous ethanol for 2 minutes in sequence;

[0017] After drying, the slides were sealed with neutral gum to obtain the second stained slides.

[0018] Preferably, the first stained slide and the second stained slide are scanned in sequence by a fully automatic cytological pathology microscope to obtain and record the results.

[0019] Preferably, the exfoliated cells include cervical fluid-based, cervical brushings, urine, pleural effusion, ascites and thyroid puncture fluid-based.

[0020] The second aspect of the present invention provides a kit for implementing the above-mentioned immunofluorescence staining and Papanicolaou staining combined detection method, the kit comprising: immunofluorescence staining reagent and EA50 staining solution.

[0021] A third aspect of the present invention provides a system for implementing the above-mentioned immunofluorescence staining and Papanicolaou staining combined detection method, the system comprising a detection module, and the detection module is used to scan the first staining slide and the second staining slide to obtain the staining result.

[0022] Preferably, the system further comprises a classification module, and the classification module is used to analyze the staining results based on a cell classification model, obtain the types of cells in the staining results, and count the number of cell types.

[0023] Compared with the prior art, the beneficial effects of the present invention are: performing two kinds of staining on the same sample piece in sequence, namely, immunofluorescence staining and Papanicolaou staining combined detection, which better improves the accuracy of cytological diagnosis results and can improve the authenticity of the results; and the two stainings will not interfere with each other. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a flow chart of the combined detection method of immunofluorescence staining and Papanicolaou staining of the present invention;

[0025] Figure 2 This is the result of immunofluorescence staining;

[0026] Figure 3 It is a partial magnified image of immunofluorescence staining;

[0027] Figure 4 This is a schematic diagram of the results of Papanicolaou staining. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0030] The present invention provides a combined detection method of immunofluorescence staining and Papanicolaou staining, such as Figure 1 As shown, the following steps are included:

[0031] Step 101: Fix the exfoliated cells or tissues to obtain a first sample slice.

[0032] The first sample slice includes a tissue slice or a cell smear. The source samples of the exfoliated cells include: cervical fluid base, cervical brushing, urine, pleural effusion, ascites and other serous cavity effusions, and thyroid and other puncture fluid bases.

[0033] Step 102: staining the first sample slice with an immunofluorescence staining reagent and sealing the slice with a cover glass to obtain a first stained slice.

[0034] Step 103: Obtain the staining result of the first staining sheet.

[0035] Step 104: After soaking the first stained slide in 90-100° C. hot water for 3-10 minutes, remove the cover glass to obtain a second sample slide.

[0036] Step 105: After staining the second sample slide with EA50 staining solution (Papanicolaou staining solution), the slide is sealed with neutral gum to obtain a second stained slide.

[0037] Step 106: Obtain the staining result of the second staining sheet.

[0038] Step 107: Mark the cell types of the staining results and construct a staining result set.

[0039] Step 108: Based on a machine learning method, the staining result set is trained to obtain a cell classification model.

[0040] The stained cells can be subjected to object detection and visual segmentation based on computer vision, and on this basis, the segmented objects can be classified.

[0041] Step 109: Analyze the staining results based on the cell classification model to obtain the type of each cell in the staining results, including diploid cells, neutrophils, inflammatory cells, lymphocytes, microorganisms, inflammatory cells, squamous cells, squamous cell carcinoma gland cells, adenocarcinoma, and impurities.

[0042] The present invention sequentially performs two stainings on the same sample sheet, namely, immunofluorescence staining and Papanicolaou staining for combined detection, which better improves the accuracy of cytological diagnosis results, and the combined detection method allows some uncertain patient specimens to be determined, which can further improve the authenticity of the results. It is of great significance in precancer screening cytological diagnosis. And the two stainings will not interfere with each other. Combined with the machine learning method, the cell types are identified and classified, the readability of the test results is improved, and important information is provided for the doctor's diagnosis.

[0043] The method of analyzing the staining according to the area where the two staining results are combined is not limited to the machine learning method. The different staining results in the same area of ​​the staining film can be manually compared to provide assistance for clinical diagnosis.

[0044] Take the exfoliated cells collected by cervical brushing as an example. The reagents used are: tap water, 95% ethanol, anhydrous ethanol, EA50, staining jar, immunofluorescence staining reagent and light-proof staining box. Among them, the immunofluorescence colorimetric reagent is a ready-to-use reagent that can be used after opening the bottle without dilution. It includes liquid A, liquid B and liquid C, of ​​which liquid A is used for staining, liquid B is used for differentiation or color separation, and liquid C is used for fixation.

[0045] Step 101: Fix the exfoliated cells to obtain a first sample slice.

[0046] Step 102: stain the first sample slice with an immunofluorescence staining reagent and seal the slice with a cover glass to obtain a first stained slice. Specifically, the following steps are included:

[0047] Step 201: Color development. Add one drop of liquid A to the fixed position of the formed component of the first sample slice, shake it slightly to cover the entire smear area, and dye for 1-3 minutes, preferably 2 minutes.

[0048] Step 202: Differentiation. Pour off the excess dye solution, add 1 drop of solution B, shake gently to cover the entire smear area, and separate the colors for 30-60 seconds.

[0049] Step 203: Sealing the slide. Remove the excess liquid by suction, and drip C solution to seal the slide to obtain the first stained slide.

[0050] Step 103: Observation: Scan and record under a fully automatic cytological pathology microscope scanner with a wavelength of 470-495 nm to obtain the staining result of the first staining slide. Figure 2 and Figure 3 The staining results are shown, and the arrows mark the neutrophils. Figure 3 This is a partial enlarged view.

[0051] Step 104: Soak in 100°C water for 5 minutes to remove the cover glass. Pure water or tap water can be used for soaking.

[0052] The method of Pap staining in step 105 includes:

[0053] Step 501: Soak in EA50 dye solution for 10 seconds. EA50 consists of red and green, and needs to be stirred before use.

[0054] Step 502: Decolorize using 9.95% ethanol, 95% ethanol, anhydrous ethanol and anhydrous ethanol in sequence for 2 minutes.

[0055] Step 503: After drying, the slide is sealed with neutral gum to obtain a second stained slide.

[0056] Step 106: Scan the second stained slide under a fully automatic cytological pathology microscope scanner and obtain the staining result. Figure 4 The second stained slide is shown with the arrows marking low-grade squamous intraepithelial lesion cells. Papanicolaou staining can also identify microorganisms, inflammatory cells, squamous cells, squamous cell carcinoma glandular cells, adenocarcinoma, etc.

[0057] In a specific test, the reagents should be replaced regularly. For example, the liquid-based thin layer preparation of exfoliated cells is stained and dehydrated with 75% alcohol, 95% alcohol, and 100% alcohol, each 500ml, and it is completely replaced every 800-1000 slides processed in a closed and continuous working environment, and a single cylinder can be used continuously. EA50 staining solution is completely replaced every 500mL in a closed and continuous working environment for 2000-2200 slides.

[0058] The present invention aims at the shortcomings of the existing single staining detection technology, and performs two stainings on the same sample piece, namely, immunofluorescence staining-Pap staining combined diagnosis, which better improves the accuracy of the precancer screening cytological diagnosis results, and the combined method can improve the authenticity of the results of some uncertain patient specimens. In addition, immunofluorescence staining and Papanicolaou staining will not interfere with the staining results.

[0059] The present invention can point to the same cell through two kinds of stained scan images, identify and jointly detect the status of the same cell, and better improve the accuracy of precancer screening cytological diagnosis results. The combined methods enable some uncertain patient specimens to be verified in multiple stained scan images, which can further improve the authenticity of the results.

[0060] The first sample piece is specifically a glass slide with exfoliated cells fixed on it. The patient specimens on the same glass slide avoid the situation that different slides (sample slices) may have inconsistent preparation results. The combined diagnosis method of immunofluorescence staining + Papanicolaou staining can better assist diagnosis and improve the accuracy and authenticity of the results. It can also reduce the amount of preparation and the screening of samples, making it less harmful to patients, less painful, and the detection method is faster. It improves the efficiency of the detection possibility for early diagnosis of cancer. Effective detection can still be performed when the number of cells is small.

[0061] The immunofluorescence staining reagent and EA50 of the present invention can adopt commercially available products. In a specific test, the immunofluorescence staining reagent uses acridine orange staining solution, which eliminates the problem of complicated solution preparation and inability to preserve. The staining steps are simplified to two-step staining with A and B staining solutions, and the staining is more convenient. The shelf life is up to 24 months. The operation is simple, the results are available in 10 minutes, the sensitivity is 85.5%, the specificity is 100%, and many morphological changes in precancerous lesions can be found. The hemoglobin in red blood cells has fluorescence inhibition and therefore does not radiate fluorescence, so the background on the entire film is black, and only the exfoliated cells that need to be observed show fluorescent reactions, which reduces the difficulty of reading the film and is more convenient for interpretation.

[0062] Each stained film of the present invention can adopt the surface scanning method to segment the visual field map, and each cell on each visual field map will be classified into diploid cells, neutrophils, inflammatory cells, lymphocytes and impurities, etc. through machine learning.

[0063] The present invention also provides a system for implementing the above-mentioned immunofluorescence staining and Papanicolaou staining combined detection method, the system comprises a detection module, and the detection module is used to scan the first staining sheet and the second staining sheet to obtain the staining result.

[0064] The system also includes a classification module, which is used to analyze the staining results based on the cell classification model, obtain the types of cells in the staining results, and count the number of cell types. A detection report can be generated based on the staining results and the analysis results.

[0065] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A combined detection method of immunofluorescence staining and Papanicolaou staining, characterized in that: The following steps are involved: Fixing the exfoliated cells to obtain a first sample slice; After staining the first sample slice with an immunofluorescence staining reagent, the slice is sealed with a cover glass to obtain a first stained slice; Obtaining a staining result of a first staining film; After soaking the first stained slide in 90-100°C hot water for 3-10 minutes, remove the cover glass to obtain a second sample slide; After staining the second sample slide with a Papanicolaou staining solution, the slide is sealed with neutral gum to obtain a second stained slide; The staining result of the second staining sheet is obtained.

2. The combined detection method of immunofluorescence staining and Papanicolaou staining according to claim 1, characterized in that: Also included is a method for identifying staining results: Mark the cell types of the staining results and construct a staining result set; Based on the machine learning method, the staining result set is trained to obtain the cell classification model; Based on the cell classification model, the staining results are analyzed to obtain the types of cells in the staining results.

3. The combined detection method of immunofluorescence staining and Papanicolaou staining according to claim 2, characterized in that: The cell types include diploid cells, neutrophils, inflammatory cells, lymphocytes, microorganisms, inflammatory cells, squamous cells, squamous cell carcinoma glandular cells and adenocarcinoma cells.

4. The combined detection method of immunofluorescence staining and Papanicolaou staining according to claim 1, characterized in that: The immunofluorescence staining reagent includes liquid A, liquid B and liquid C, and the method for staining the first sample sheet includes: Add solution A dropwise onto the visible components of the first sample slide, shake gently to cover the entire smear area, and stain for 1-3 minutes; Pour off excess dye, add solution B, shake gently to cover the entire smear area, and separate the colors for 30-60 seconds; Remove excess residual liquid by suction, add liquid C to seal the slide, and obtain the first stained slide.

5. The combined detection method of immunofluorescence staining and Papanicolaou staining according to claim 1, characterized in that: The method of staining the second sample slide with the Papanicolaou staining solution includes: Soak in EA50 staining solution for 10 seconds; Decolorize with 9.95% ethanol, 95% ethanol, anhydrous ethanol, and anhydrous ethanol for 2 minutes in sequence; After drying, the slides were sealed with neutral gum to obtain the second stained slides.

6. The combined detection method of immunofluorescence staining and Papanicolaou staining according to claim 1, characterized in that: The first staining slide and the second staining slide are scanned in sequence by a fully automatic cytological pathology microscope to obtain and record the results.

7. The combined detection method of immunofluorescence staining and Papanicolaou staining according to claim 1, characterized in that: The exfoliated cells include cervical liquid base, cervical brushing material, urine, pleural effusion, ascites and thyroid puncture liquid base.

8. A kit, characterized in that: Used to implement the combined detection method of immunofluorescence staining and Papanicolaou staining as described in any one of claims 1 to 7, the kit comprises: immunofluorescence staining reagent and EA50 staining solution.

9. A system, characterized in that: Used to implement the combined detection method of immunofluorescence staining and Papanicolaou staining as described in any one of claims 1 to 7, the system comprises a detection module, The detection module is used to scan the first stained film and the second stained film to obtain a staining result.

10. The system according to claim 9, characterized in that Also includes classification modules, The classification module is used to analyze the staining results based on the cell classification model to obtain the types of cells in the staining results.

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