A pathological cytology blood removal kit and its usage method

Abstract

The present invention relates to the technical field of pathological cell analysis, and in particular to a blood removal kit for pathological cytology and its usage method, which includes a blood removal reagent and a cell anti - detachment glass slide; the blood removal reagent includes alcohols, glacial acetic acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, a mucus softening agent, and deionized water. The reagents used are low - cost and are all common chemical reagents. The operation is simple and easy to understand, and it is applicable to various pathological cell blood - containing samples. When preparing pathological cell smears using the kit provided by the present invention, for blood - containing samples, it can effectively remove the interference of red blood cells and mucus components, enabling better cell enrichment, and maintaining the original morphology of diseased cells and normal cells. Most cells can be preserved on the glass slide during slide preparation, reducing the probability of slide preparation failure caused by cell detachment, improving the excellent slide rate of pathological cell slides, and enhancing the accuracy of pathological cytology diagnosis.

Description

Technical Field

[0001] The present invention relates to the technical field of pathological cell analysis, and in particular to a pathological cytology blood removal kit and a method for using the same. Background Art

[0002] Clinical cytology samples submitted for examination often form bloody samples due to damage to the sampling site or invasion of blood vessels by lesions. Most of them appear in malignant pleural effusion, pericardial effusion, and cytology samples obtained by puncture sampling. Sometimes, bleeding may also occur during the lavage sampling process in parts such as the bronchi and alveoli, forming bloody samples. Due to the easy coagulation property of blood, there may also be some jelly-like substances in a small amount of samples, which will seriously affect the quality of pathological cell smears and increase the difficulty of interpreting pathological cells.

[0003] The above-mentioned difficulties in interpretation are mainly manifested as follows: (1) In terms of centrifugal enrichment of cells in the sample: The presence of a large number of blood cells and some jelly-like substances affects the discrimination of the effective cell layer for observing cell precipitation. When the sample is thick with blood, there will be no stratification state, increasing the difficulty of slide preparation, and it is impossible to ensure whether effective cells can be accurately extracted, reducing the cell content on the slide; (2) In terms of microscopic cell interpretation: Since a large amount of hemoglobin and blood cells adhere to the slide under the microscope field of view, they will cover some pathological cells, reducing the number of pathological cells under the microscope and making it impossible for the interpreter to clearly observe the cell morphology, thus affecting the interpretation. Therefore, removing the interference of red blood cells is the key to making high-quality pathological cell smears.

[0004] The existing methods for removing blood from pathological cell samples mainly include the following several methods: 1. High-concentration glacial acetic acid-based cell cleaning solution; 2. A liquid mixture of ethanol or methanol at a certain concentration; 3. Commercial hemolytic agents with potassium cyanide or hemolytic agents containing ammonium chloride. Among the above three methods, in method 1, while lysing red blood cells with a high-concentration glacial acetic acid-based cell cleaning solution, some diseased cells will also be lost, which is likely to cause missed diagnosis; method 2 uses the principle of osmotic pressure to lyse red blood cells, and low-concentration alcohols cannot better fix the cell morphology, causing them to degenerate and affecting the diagnosis; method 3, the commercial hemolytic agent is relatively expensive, and the potassium cyanide hemolytic agent contains highly toxic potassium cyanide components, which pose a threat to both the operator and the environment; while the ammonium chloride hemolytic agent needs to be diluted immediately before use and cannot be stored for a long time. After blood removal, the cell structure is clear, but there are still many red blood cell shadows in the background. Especially when the sample is thick with blood, blood removal is not thorough, and it has an agglomerating effect on cells, making the cells easy to overlap and affecting the observation.

[0005] In addition, another key point in making high-quality pathological cell smears is to prevent pathological cells from falling off the glass slides during slide preparation. In recent years, there are many brands and types of anti-drop glass slides dedicated to pathology, which are mainly applicable to special staining, immunohistochemistry, frozen section, etc. For the treatment of pathological cells, the most commonly used method at present is liquid-based thin-layer preparation, which is not standardized in the production of conventional pathological cell smears and spin smears. The phenomenon of cell shedding is widespread. Especially for samples treated with blood removal, the cell adhesion decreases and the shedding is more serious, which brings great troubles to clinical pathological diagnosis, delays the diagnosis time, and even has the risk of missed diagnosis and misdiagnosis.

[0006] In view of the above problems, developing a new type of blood removal kit for pathological cytology and its use method to improve the accuracy of pathological cytology diagnosis is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0007] The first object of the present invention is to provide a blood removal kit for pathological cytology, which ensures the consistency between the microscopic field of view and the submitted sample, and improves the accuracy of pathological cytology diagnosis;

[0008] The second object of the present invention is to provide a use method of the blood removal kit for pathological cytology, which standardizes the operation process of pathological cell detection and improves the accuracy of pathological cytology diagnosis.

[0009] The present invention provides a blood removal kit for pathological cytology, including a blood removal reagent and a cell anti-drop glass slide; the blood removal reagent includes alcohols, glacial acetic acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, a mucus softening agent and deionized water; and the amounts of glacial acetic acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and the mucus softening agent corresponding to every 25-30 mL of alcohols are 4-8 mL, 0.02-0.025 g, 0.1-0.15 g, 0.6-0.8 g and 0.1-0.15 g respectively.

[0010] The blood removal kit for pathological cytology of the present invention includes a blood removal reagent and a cell anti - detachment glass slide. Among them, the reagents used in the blood removal reagent are of low cost and are all common chemical reagents. Among them, alcohols account for 25% - 30% of the blood removal reagent. At this time, the solution is in a hypotonic state and the tension on the cells is increased, which can lyse anucleated red blood cells. At the same time, it can fix the albumin of other nucleated cells, so that the nucleated cells maintain their general morphology. When the concentration of alcohols is lower than this concentration, the nucleated cells become swollen and degenerate. When the concentration of alcohols is higher than this concentration, hemoglobin denatures and is difficult to redissolve, producing a large amount of bloody sediment. The content of glacial acetic acid accounts for 4% - 8% of the blood removal reagent. This concentration of glacial acetic acid can both lyse red blood cells and fix the nuclear protein of diseased cells, and is not affected by the hypotonic environment and tension, so that the cells maintain their original morphology. When the concentration of glacial acetic acid is lower than the lower limit concentration, it cannot effectively fix the nuclear protein. When the concentration of glacial acetic acid is higher than the upper limit concentration, the cells will expand and change their normal structure. Sodium dihydrogen phosphate and disodium hydrogen phosphate form a buffer system, making the whole blood removal reagent weakly acidic with a wide buffer range, which can be suitable for a variety of pathological cell samples. And the mucus softener N - acetyl can remove the mucus present in the sample, so that all diseased cells are released.

[0011] Therefore, under the interaction of the above - mentioned components of the blood removal reagent of the present invention, it can process a variety of bloody samples, and can protect the effective cell components while removing blood, so as to preserve most of the cells in their original state on the glass slide. When making a pathological cell smear with the kit provided by the present invention, for bloody samples, it can effectively remove the interference of red blood cells and mucus components, make the cells better enriched, and maintain the original morphology of diseased cells and normal cells. Most of the cells can be preserved on the glass slide during specimen preparation, reducing the probability of specimen preparation failure caused by cell detachment and improving the excellent - specimen rate of pathological cell slides.

[0012] Preferably as this technical solution, the alcohols include any one of methanol and ethanol or a mixture composed of the two in any proportion, and the mass concentration of the methanol and the ethanol is 95 - 100%.

[0013] Preferably as this technical solution, the mucus softener is N - acetyl - L - cysteine.

[0014] N - acetyl - L - cysteine, the mucus softener in the present invention, accounts for 0.1% - 0.15% of the blood removal reagent. Its function is to remove the mucus present in the sample, so that all diseased cells are released, thus facilitating the enrichment of diseased cells. And its concentration increases as the concentration of glacial acetic acid decreases, because glacial acetic acid also has a part of the function of dissolving mucus.

[0015] Preferably as this technical solution, the cell anti - detachment glass slide is obtained by processing a glass slide through the following steps:

[0016] S1. Immerse the glass slide in the cleaning solution for soaking and cleaning, fish it out, and then wash it with clean water and drain it.

[0017] S2. Immerse the glass slide obtained in step S1 in a saturated solution of concentrated sulfuric acid and potassium dichromate for 10 - 20 h, fish it out, wash it with clean water and drain it, then immerse it in a 95% ethanol solution for 1 - 3 h, take it out and air-dry it dust-free.

[0018] S3. Drop the anti - detachment working solution on the surface of the glass slide obtained in step S2, cover it with another glass slide, and use the pulling method to separate the two glass slides, air-dry them in a cool and dust-free place, and repeat the above steps once to obtain the cell anti - detachment glass slide.

[0019] Among them, step 1 specifically includes: adding enzyme - containing washing powder and purified water in a ratio of 1:50, adding the washing powder to hot water to prepare the cleaning solution, immersing the ordinary glass slide in the cleaning solution, turning it several times during this period, when the water temperature is touchable by hand, fish out the glass slide, rinse it several times under running water until there is no foam and no mark residue, wash it with clean water and drain it. To remove impurities, dust and grease on the surface of the glass slide and reduce the surface roughness.

[0020] Step S2 specifically includes: immersing the glass slide in the saturated solution of concentrated sulfuric acid and potassium dichromate overnight, carefully fish it out and then put it into clean water to wash it thoroughly and drain it, finally immerse it in a 95% ethanol solution for 2 h, take it out and air-dry it dust-free. Check the cleaning degree. Let the surface of the glass slide be in a hydrophilic state. The presence of a retained water film indicates that it is clean. The purpose of pickling is to further remove surface residual impurities and various adsorbed pollutants and further reduce the roughness.

[0021] Step S3 specifically includes: preparing the anti - detachment working solution by mixing polylysine and double - distilled water in a ratio of 5:5 or 6:4, dropping the anti - detachment working solution on the surface of the glass slide, covering it with another glass slide, allowing the anti - detachment working solution to naturally and evenly spread over the entire surface, using the pulling method to separate the two glass slides, placing them in a cool and dust-free place to dry naturally, and treating the glass slide again in the same way to further improve the treatment effect of the glass slide. In addition, during the anti - detachment treatment, in order to distinguish the anti - detachment treatment surface during use, the rough surface and the anti - detachment treatment surface can be written on the same side of the slide head.

[0022] Preferably in this technical solution, the cleaning solution is prepared from enzyme - containing washing powder and purified water with a mass ratio of 1:(45 - 55), and the temperature of the purified water is 60 - 90 °C.

[0023] Preferably in this technical solution, the anti - detachment working solution is prepared from polylysine and double - distilled water with a mass ratio of (1 - 1.5):1.

[0024] The present invention also discloses a method for using the above-mentioned pathological cytology blood removal kit, which should also belong to the protection scope of the present invention. The specific steps are as follows:

[0025] S1. Add a blood removal reagent to the pretreated bloody sample, and perform shaking and centrifugation in sequence to obtain cell precipitates.

[0026] S2. Aspirate the cell precipitates onto the anti - detachment treated surface of a cell anti - detachment glass slide, and perform air - drying, fixation, staining, and mounting in sequence.

[0027] Preferably, in step S1, the pretreatment method of the bloody sample is: centrifuge the bloody sample at 1800 - 2200 rpm for 4 - 6 min, remove the supernatant to obtain the pretreated bloody sample.

[0028] Preferably, in step S1, the mass ratio of the pretreated bloody sample to the blood removal reagent is 1:(5 - 6).

[0029] Preferably, during the shaking, control the rotation speed at 1400 - 1600 rpm and the time at 1 - 2 min; during the centrifugation, control the rotation speed at 1800 - 2200 rpm and the time at 4 - 6 min.

[0030] That is, the specific method for using the pathological cytology blood removal kit of the present invention is:

[0031] (1) Transfer the bloody sample of a volume according to the actual situation into a 10 ml - 50 ml centrifuge tube, centrifuge at 2000 rpm for 5 min to enrich the cells, remove the supernatant, add the blood removal reagent to the precipitate at a ratio of 1:(5 - 6), shake at 1500 rpm for 1 - 2 min, and then centrifuge at 2000 rpm for 5 min to enrich the cells and remove the supernatant; if the blood removal is not complete, the previous steps can be repeated.

[0032] (2) After the blood removal is completed, aspirate the upper layer of the cell precipitate with a pipette or a micropipette, take a volume the size of a soybean and drop it on the anti - detachment treated surface of the cell anti - detachment glass slide, cover it with the glue - coated surface of another glass slide, let the cell precipitate solution naturally and evenly spread over the entire surface, gently pull the glass slides to separate them, air - dry and place them in a separate cylinder containing 95% ethanol for fixation for more than 15 min, and then perform staining and mounting.

[0033] In summary, the method for using the pathological cytology blood removal kit of the present invention is standardized, efficient, non - toxic, and can ensure the quality of the preparation, making the subsequent pathological cytology diagnosis and interpretation easier and more accurate.

[0034] The pathological cytology blood removal kit of the present invention has at least the following technical effects:

[0035] 1. The blood-removing kit for pathological cytology of the present invention includes a blood-removing reagent and a cell anti-drop glass slide. Among them, the used blood-removing reagent includes alcohols, glacial acetic acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, and a mucus softening agent. The used reagents are low-cost and are all common chemical reagents. The operation is simple and easy to understand. It is applicable to various pathological cell blood samples. While removing blood, it also protects the effective cell components, and most cells can be preserved on the glass slide in their original state, without cell shedding caused by factors such as immediate fixation, water washing, and staining, which may contaminate the reagent or other glass slides, resulting in missed diagnosis or misdiagnosis. Therefore, the blood-removing reagent of the present invention ensures the consistency between the microscopic field of view and the submitted sample, and improves the accuracy of pathological cytological diagnosis;

[0036] 2. The cell anti-drop glass slide in the blood-removing kit for pathological cytology of the present invention is obtained by treating a glass slide through a special method. During the treatment process, the ordinary glass slide is successively treated with a cleaning solution, a saturated solution of concentrated sulfuric acid and potassium dichromate, and an anti-drop solution. When this cell anti-drop glass slide is used for pathological cell analysis, the phenomenon of cell shedding is significantly improved, solving the problem of missed diagnosis or misdiagnosis caused by cell shedding, and improving the accuracy of pathological cytological diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0038] Figure 1 It is a light microscope image of a blood-containing pleural effusion cell preparation without treatment in Control Example 1;

[0039] Figure 2 It is a light microscope image of a blood-containing pleural effusion cell preparation treated with the blood-removing kit for pathological cytology of the present invention in Control Example 2;

[0040] Figure 3 It is a light microscope image of a blood-containing pleural effusion cell preparation (using a cell anti-drop glass slide) treated with a high-concentration glacial acetic acid-based cell cleaning solution in Control Example 3;

[0041] Figure 4 It is a light microscope image of a blood-containing pleural effusion cell preparation (using a cell anti-drop glass slide) treated with a commercial ammonium chloride hemolytic agent in Control Example 4;

[0042] Figure 5 It is a light microscope image of a blood-containing pleural effusion cell preparation (using a cell anti-drop glass slide) treated with a 30% ethanol solvent in Control Example 5;

[0043] Figure 6Light microscope image of the cell preparation of bloody pleural effusion treated with the pathological cytology blood removal kit of the present invention for Comparative Example 6 (using a conventional hydrophilic HE glass slide);

[0044] Figure 7 Light microscope image of the cell preparation of bloody pleural effusion treated with the pathological cytology blood removal kit of the present invention for Comparative Example 7 (using a conventional hydrophobic HE glass slide);

[0045] Figure 8 Light microscope image of the cell preparation of bloody pleural effusion treated with the pathological cytology blood removal kit of the present invention for Comparative Example 8 (using a liquid-based cell glass slide);

[0046] Figure 9 Light microscope image of the cell preparation of bloody pleural effusion treated with the pathological cytology blood removal kit of the present invention for Comparative Example 9 (using a positively charged immunohistochemical non-falling-off glass slide). Detailed implementation manners

[0047] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0048] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary implementation manners according to the present application. As used herein, unless the context clearly indicates otherwise, the singular forms also include the plural forms. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0049] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0050] Example 1

[0051] Blood removal reagent ratio: 250 ml of ethanol, 80 ml of glacial acetic acid, 0.2 g of sodium dihydrogen phosphate, 1 g of disodium hydrogen phosphate, 6 g of sodium chloride, 1 g of N-acetyl-L-cysteine, and made up to 1000 ml with deionized water.

[0052] Preparation method of the blood removal reagent: After weighing and measuring according to the above components, stir and dissolve.

[0053] Cell non-falling-off glass slide treatment method:

[0054] S1. Slide cleaning: Add enzyme-containing washing powder to 80°C hot water to prepare a cleaning solution. Among them, the mass ratio of enzyme-containing washing powder to purified water is 1:50. Immerse ordinary glass slides in the cleaning solution and turn them several times. When the water temperature is touchable by hand, take out the glass slides, wash them with clean water and drain them.

[0055] S2. Acid treatment: Immerse the glass slides in a saturated solution of concentrated sulfuric acid and potassium dichromate for 10 h, carefully take them out and then fully wash them with clean water and drain them. Finally, immerse them in a 95% ethanol solution for 2 h, take them out and air-dry them in a dust-free environment.

[0056] S3. Anti-detachment treatment: Prepare an anti-detachment working solution according to the mass ratio of polylysine to double-distilled water of 5:5. Drop the anti-detachment working solution on the surface of the glass slide, cover it with another glass slide, and let the anti-detachment working solution naturally and evenly spread over the entire surface. Separate the two glass slides by the pulling method, place them in a cool and dust-free place to dry naturally, and treat the glass slides again in the same way to obtain cell anti-detachment glass slides.

[0057] Usage method of the blood removal kit for pathological cytology of the present invention:

[0058] S1. Transfer 50 ml of bloody pleural effusion into a centrifuge tube, centrifuge at 2000 rpm for 5 min to enrich cells, remove the supernatant, add 25 ml of blood removal reagent to 5 ml of the precipitate, shake at 1500 rpm for 1 - 2 min, and then centrifuge at 2000 rpm for 5 min to enrich cells, remove the supernatant; suck out the upper layer of the cell precipitate with a pipette or micropipette, take a volume the size of a soybean and drop it on the anti-detachment working solution-treated surface of a special cell anti-detachment glass slide, perform pulling, air-dry it and place it in a separate cylinder containing 95% ethanol to fix it for more than 15 min, and then stain and cover the slide.

[0059] Observation under the microscope shows that the background blood removal is relatively clean and simple, the cells are rich and clear, and the structure is complete, which is convenient for diagnosis.

[0060] Example 2

[0061] Blood removal reagent formulation: 270 ml of ethanol, 50 ml of glacial acetic acid, 0.22 g of sodium dihydrogen phosphate, 1.2 g of disodium hydrogen phosphate, 7 g of sodium chloride, 1.2 g of N-acetyl-L-cysteine, and make up to 1000 ml with deionized water.

[0062] The preparation method of the blood removal reagent is the same as that in Example 1.

[0063] The treatment method of the cell anti-detachment glass slide is the same as that in Example 1.

[0064] Usage method of the blood removal kit for pathological cytology of the present invention:

[0065] Transfer 30 ml of bloody ascites sample into a centrifuge tube, centrifuge at 2000 rpm for 5 min to enrich cells, remove the supernatant, add 20 ml of blood removal reagent to the 4 ml precipitate, shake at 1500 rpm for 1 - 2 min, then centrifuge at 2000 rpm for 5 min to enrich cells, and remove the supernatant; suck out the upper layer of the cell precipitate with a pipette or micropipette, take a sample the size of a soybean and drop it on the anti - detachment working solution treatment surface of the cell anti - detachment slide, perform spreading, air - dry it, and place it in a separate cylinder filled with 95% ethanol for fixation for more than 15 min, and then stain and cover - slip.

[0066] Observed under the microscope, the background is completely de - blooded, the effective cells are prominent, the structure is clear and complete, which is convenient for diagnosis.

[0067] Example 3

[0068] Blood removal reagent formulation: 280 ml of ethanol, 80 ml of glacial acetic acid, 0.25 g of sodium dihydrogen phosphate, 1.5 g of disodium hydrogen phosphate, 8 g of sodium chloride, 1 g of N - acetyl - L - cysteine, made up to 1000 ml with deionized water.

[0069] The preparation method of the blood removal reagent is the same as that in Example 1.

[0070] The treatment method of the cell anti - detachment slide is the same as that in Example 1.

[0071] The usage method of the blood removal kit for pathological cytology of the present invention:

[0072] Transfer 20 ml of bloody pericardial effusion sample into a centrifuge tube, centrifuge at 2000 rpm for 5 min to enrich cells, remove the supernatant, add 30 ml of blood removal reagent to the 6 ml precipitate, shake at 1500 rpm for 1 - 2 min, then centrifuge at 2000 rpm for 5 min to enrich cells, and remove the supernatant; because the blood removal is not complete, repeat the above operation and add 20 ml of blood removal reagent. After blood removal, suck out the upper layer of the cell precipitate with a pipette or micropipette, take a sample the size of a soybean and drop it on the anti - detachment working solution treatment surface of the cell anti - detachment slide, perform spreading, air - dry it, and place it in a separate cylinder filled with 95% ethanol for fixation for more than 15 min, and then stain and cover - slip.

[0073] Observed under the microscope, the background is relatively clean after blood removal, only a small amount of hemoglobin precipitate remains, the number of cells is abundant, the structure is complete, the cell nucleus and cytoplasm are clear, which is convenient for diagnosis.

[0074] Example 4

[0075] Blood removal reagent formulation: 300 ml of methanol, 60 ml of glacial acetic acid, 0.2 g of sodium dihydrogen phosphate, 1.2 g of disodium hydrogen phosphate, 6 g of sodium chloride, 1.2 g of N - acetyl - L - cysteine, made up to 1000 ml with deionized water.

[0076] The preparation method of the blood removal reagent is the same as that in Example 1.

[0077] In the treatment method of the cell anti - detachment slide, the mass ratio of polylysine to double - distilled water is 6:4, and the others are the same as in Example 1.

[0078] The usage method of the pathological cytology blood removal kit of the present invention:

[0079] Transfer 10 ml of the bloody lymph node rinse sample into a centrifuge tube, centrifuge at 2000 rpm for 5 min to enrich the cells, remove the supernatant, add 18 ml of the blood removal reagent to 3 ml of the precipitate, shake at 1500 rpm for 1 - 2 min, then centrifuge at 2000 rpm for 5 min to enrich the cells, and remove the supernatant; suck out the cell precipitate with a pipette or pipettor, drop it on the anti - detachment working fluid treatment surface of the cell anti - detachment slide, perform spreading, air - dry it, and put it into a separate cylinder filled with 95% ethanol for fixation for more than 15 min, and then stain and cover - slip.

[0080] Observed under the microscope, the background is relatively clean, lymphocytes are abundant, the effective cell structure is clear and complete, which is convenient for observation.

[0081] Example 5

[0082] The formulation of the blood removal reagent: 270 ml of methanol, 50 ml of glacial acetic acid, 0.22 g of sodium dihydrogen phosphate, 1.3 g of disodium hydrogen phosphate, 7 g of sodium chloride, 1.3 g of N - acetyl - L - cysteine, and made up to 1000 ml with deionized water.

[0083] The preparation method of the blood removal reagent is the same as that in Example 1.

[0084] The treatment method of the cell anti - detachment slide is the same as that in Example 4.

[0085] The usage method of the pathological cytology blood removal kit of the present invention:

[0086] Transfer 10 ml of the bloody alveolar lavage fluid sample into a centrifuge tube, centrifuge at 2000 rpm for 5 min to enrich the cells, remove the supernatant, add 10 ml of the blood removal reagent to 2 ml of the precipitate, shake at 1500 rpm for 1 - 2 min, then centrifuge at 2000 rpm for 5 min to enrich the cells, and remove the supernatant; suck out the cell precipitate with a pipette or pipettor, drop it on the anti - detachment working fluid treatment surface of the cell anti - detachment slide, perform spreading, air - dry it, and put it into a separate cylinder filled with 95% ethanol for fixation for more than 15 min, and then stain and cover - slip.

[0087] Observed under the microscope, the background is relatively clean, cells are abundant, the structure is complete and clear, there is no mucus substance blocking the field of view, which is convenient for observation.

[0088] To further study the blood removal effect of the blood removal reagent in this application, the hemorrhagic pleural effusion was divided into 5 parts, each part included 30 ml of sample, and they were respectively denoted as sample 1, sample 2, sample 3, sample 4 and sample 5, and the following control experiments were carried out.

[0089] Control Example 1

[0090] The sample 1 was mixed, centrifuged and concentrated according to Example 1, the upper layer was taken and directly spread on a cell adhesion slide, air-dried and fixed for more than 15 min, and then stained and sealed. Microscopic examination was carried out to obtain Figure 1 The slide preparation effect.

[0091] It can be seen from Figure 1 that when preparing slides with untreated hemorrhagic pleural effusion cells, even using the cell adhesion slide of the present invention, a large number of red blood cells still block the field of view of the effective cells, the structure is blurred, and it is difficult to diagnose.

[0092] Control Example 2

[0093] The sample 2 was mixed, centrifuged and concentrated according to Example 1, 15 ml of the blood removal reagent of the present invention was added, and it was processed according to the method of Example 1, the cell precipitate was extracted, directly spread on a cell adhesion slide, air-dried and fixed for more than 15 min, and then stained and sealed. Microscopic examination was carried out to obtain Figure 2 The slide preparation effect.

[0094] It can be seen from Figure 2 that when preparing slides with hemorrhagic pleural effusion cells treated with the pathological cytology blood removal kit of the present invention. The light microscope image has a clean background, rich cells, clear structure, and is easy to diagnose.

[0095] Control Example 3

[0096] The sample 3 was mixed, centrifuged and concentrated according to Example 1, treated with 15 ml of high-concentration glacial acetic acid liquid-based cell cleaning solution, shaken at 1500 rpm for 2 min, and then centrifuged at 2000 rpm for 5 min, the cell precipitate was extracted, directly spread on a cell adhesion slide, air-dried and fixed for more than 15 min, and then stained and sealed. Microscopic examination was carried out to obtain Figure 3 The slide preparation effect.

[0097] It can be seen from Figure 3 that when treating hemorrhagic pleural effusion cells with the high-concentration glacial acetic acid liquid-based cell cleaning solution in the prior art and using the cell adhesion slide of the present invention for slide preparation. The light microscope image has a clean background and a large amount of cells, but due to the too high concentration of glacial acetic acid, the cell plasma boundary is unclear and there are many naked nuclei.

[0098] Control Example 4

[0099] The sample 4 was mixed, centrifuged and concentrated according to Example 1, treated with 15 ml of commercial ammonium chloride lysing agent, shaken manually several times, allowed to stand for 5 min, centrifuged at 2000 rpm for 5 min, the cell precipitate was extracted, directly smeared using a cell adhesion prevention slide, air-dried and fixed for more than 15 min, and then stained and mounted. Microscopic examination gave Figure 4 the preparation effect.

[0100] It can be seen from Figure 4 that when treating bloody pleural effusion cells with the commercial ammonium chloride lysing agent in the prior art and using the cell adhesion prevention slide of the present invention for preparation, there are still many red blood cell shadows in the light microscope image background, the cell structure is complete, but the cells are relatively aggregated, and some cell nuclei are not clear, which will cause trouble for diagnosis.

[0101] Control Example 5

[0102] The sample 5 was mixed, centrifuged and concentrated according to Example 1, treated with 15 ml of 30% ethanol solvent, shaken manually more than a dozen times, centrifuged at 2000 rpm for 5 min, the cell precipitate was extracted, directly smeared using a cell adhesion prevention slide, air-dried and fixed for more than 15 min, and then stained and mounted. Microscopic examination gave Figure 5 the preparation effect.

[0103] It can be seen from Figure 5 that when treating bloody pleural effusion cells with 30% ethanol solvent in the prior art and using the cell adhesion prevention slide of the present invention for preparation, the cell quantity in the light microscope image is acceptable, but the nucleoli of some cell nuclei degenerate, and there is a phenomenon that the nuclear membrane boundary is unclear.

[0104] Control Example 6

[0105] The cell precipitate extracted from sample 2 was directly smeared using a conventional hydrophilic HE slide, air-dried and fixed for more than 15 min, and then stained and mounted. Microscopic examination gave Figure 6 the preparation effect.

[0106] It can be seen from Figure 6 that when treating bloody pleural effusion cells with the pathological cytology blood removal kit of the present invention, but using a conventional hydrophilic HE slide in the current technology for preparation, the background of the light microscope image is clean, the cell structure is clear, but the cells fall off seriously and the cell quantity is small, which affects the diagnosis.

[0107] Control Example 7

[0108] The cell precipitate extracted from sample 2 was directly smeared using a conventional hydrophobic HE slide, air-dried and fixed for more than 15 min, and then stained and mounted. Microscopic examination gave Figure 7 the preparation effect.

[0109] It can be seen from Figure 7It can be seen that the blood-containing pleural effusion cells treated with the pathological cytology blood-removing kit of the present invention are prepared using a conventional hydrophobic HE slide in the prior art. The background of the light microscope image is clean, the cell structure is clear, but the cells fall off severely, which does not match the original cell quantity and affects the diagnosis.

[0110] Control Example 8

[0111] The cell precipitate extracted from Sample 2 was directly drawn on a liquid-based cell slide, air-dried and fixed for more than 15 minutes, and then stained and sealed. Microscopic examination was performed to obtain Figure 8 the slide preparation effect.

[0112] It can be seen from Figure 8 that the blood-containing pleural effusion cells treated with the pathological cytology blood-removing kit of the present invention are prepared using a liquid-based cell slide in the prior art. The adhesive layer on the surface of the light microscope slide reacts with the components in the removed blood, and a large area of cells fall off, leaving only a very small amount of cells.

[0113] Control Example 9

[0114] The cell precipitate extracted from Sample 2 was directly drawn on a positively charged immunohistochemical anti-detachment slide, air-dried and fixed for more than 15 minutes, and then stained and sealed. Microscopic examination was performed to obtain Figure 9 the slide preparation effect.

[0115] It can be seen from Figure 9 that the blood-containing pleural effusion cells treated with the pathological cytology blood-removing kit of the present invention are prepared using a positively charged immunohistochemical anti-detachment slide in the prior art. The background of the light microscope image is clean, the cell structure is clear, and the cell quantity is acceptable, but compared with Figure 2 there are still significant differences.

[0116] In summary, the blood-removing reagent in the pathological cytology blood-removing kit of the present invention protects the effective cell components while removing blood, and can preserve most cells in their original state on the slide. Moreover, the specially treated cell anti-detachment slide can effectively prevent cells from falling off due to factors such as fixation, water washing, and staining. Therefore, the pathological cytology blood-removing kit of the present invention improves the accuracy of pathological cytology diagnosis.

[0117] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pathological cytology blood removal kit, characterized in that, Comprising a blood removal reagent and a cell anti - detachment slide; The blood removal reagent is composed of alcohol, glacial acetic acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, a mucus softening agent and deionized water; and for every 25 - 30 mL of alcohol, the amounts of glacial acetic acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and the mucus softening agent are 4 - 8 mL, 0.02 - 0.025 g, 0.1 - 0.15 g, 0.6 - 0.8 g and 0.1 - 0.15 g respectively; The alcohol includes any one of methanol and ethanol or a mixture composed of the two in any proportion, and the mass concentration of the methanol and the ethanol is 95 - 100%; The mucus softening agent is N - acetyl - L - cysteine; The cell anti - detachment slide is obtained by treating a glass slide through the following steps: S1. Immerse the glass slide in a cleaning solution for soaking and cleaning, fish it out and then wash it with clean water and drain; S2. Immerse the glass slide obtained in step S1 in a saturated solution of concentrated sulfuric acid and potassium dichromate for 10 - 20 h, fish it out and then wash it with clean water and drain, and then immerse it in a 95% ethanol solution for 1 - 3 h, take it out and air - dry it in a dust - proof place; S3. Drop the anti - detachment working solution on the surface of the glass slide obtained in step S2, cover it with another glass slide, and use the pulling - sheet method to separate the two glass slides, dry it in a cool and dust - proof place, and repeat the above steps once to obtain the cell anti - detachment slide; The cleaning solution is prepared from enzyme - added washing powder and purified water with a mass ratio of 1:(45 - 55), and the temperature of the purified water is 60 - 90 °C; The anti - detachment working solution is prepared from polylysine and double - distilled water with a mass ratio of (1 - 1.5):

1.

2. The method for using the pathological cytology blood removal kit according to claim 1, characterized in that, Including the following steps: S1. Add the blood removal reagent to the pretreated blood - containing sample, and perform shaking and centrifugation in sequence to obtain cell precipitate; S2. Aspirate the cell precipitate onto the anti - detachment treatment surface of the cell anti - detachment slide, and perform air - drying, fixation, staining and sealing in sequence.

3. The usage method according to claim 2, characterized in that, In step S1, the pretreatment method of the blood - containing sample is: centrifuge the blood - containing sample at 1800 - 2200 rpm for 4 - 6 min, remove the supernatant to obtain the pretreated blood - containing sample.

4. The usage method according to claim 2, wherein In step S1, the mass ratio of the pretreated blood - containing sample to the blood removal reagent is 1:(5 - 6).

5. The usage method according to claim 2, characterized in that, When shaking, control the rotation speed to be 1400 - 1600 rpm and the time to be 1 - 2 min; When centrifuging, control the rotation speed to be 1800 - 2200 rpm and the time to be 4 - 6 min.

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