High-adaptation, high-definition chromosome preparation method for bone marrow cells of malignant hematopathy

Abstract

This invention belongs to the field of biomedical technology. Specifically, it discloses a highly adaptable and high-resolution method for preparing chromosome slides from bone marrow cells of malignant hematological diseases. This invention employs a dual-concentration colchicine treatment, hypotonic potassium chloride solution, two-step fixation, low-position slide preparation, and precise banding staining, achieving a comprehensive improvement in chromosome specimen quality. It ensures a sufficient number of metaphase cells; the optimized hypotonic solution and two-step fixation ensure sufficient dispersion and complete preservation of chromosomes in each phase; the low-position slide preparation ensures uniform distribution of chromosomes on the slide; and the precise banding staining parameters ensure that the bands of each chromosome are clearly distinguishable.

Description

Technical Field

[0001] This invention relates to the field of biomedical cytogenetics detection technology, specifically to a method for preparing chromosome slides of bone marrow cells from malignant hematological diseases with high adaptability and high resolution. Background Technology

[0002] Bone marrow cell karyotype analysis is a crucial tool in the cytogenetic study of hematologic diseases, particularly leukemia. Cytogenetic analysis has become one of the core technologies for leukemia diagnosis, subtyping, prognostic assessment, and treatment monitoring. Karyotype analysis not only detects clonal chromosomal abnormalities, providing key evidence for the diagnosis and differential diagnosis of leukemia and myelodysplastic syndromes (MDS), but also identifies AML and ALL and their subtypes through specific chromosomal rearrangements. Furthermore, chromosomal aberrations serve as important indicators for monitoring acute leukemia remission, relapse, and blast crisis in chronic myeloid leukemia (CML). Sex chromosome markers can be used to verify the success of allogeneic bone marrow transplantation, and karyotype is an independent prognostic indicator that directly influences treatment selection. Cloning of chromosomal translocation breakpoints has also led to the discovery of a series of important genes related to leukemia, providing crucial clues for molecular research and minimal residual disease detection.

[0003] Currently, there are four main methods for preparing bone marrow cell chromosomes: the direct method, the short-term culture method, the synchronous method, and the high-metaphase method. The direct method involves immediately processing bone marrow samples after extraction without prior culture before slide preparation. This method was once widely recommended by scholars, who believed it could reflect the true karyotype of cells in vivo and avoid selective cell growth that might occur during culture. However, the direct method produces a smaller number of mitotic figures, resulting in lower chromosome quality; chromosomes often appear short, forked, or even hairy, which is detrimental to various banding treatments and severely limits the accuracy of karyotype analysis. The short-term culture method involves seeding bone marrow cells at a specific density into a culture medium after nucleated cell counting, and then harvesting the cells for slide preparation after 24 or 48 hours of culture. Short-term culture increases the proliferation rate of leukemia cells while decreasing the proliferation rate of normal cells, thus significantly increasing the detection rate of abnormal karyotypes. The synchronization method involves treating cells with methotrexate or fluorodeoxyuridine for 17 hours to synchronize them, followed by short-term treatment with colchicine to obtain a large number of mitotic phases in late prophase, prometaphase, early metaphase, and metaphase, significantly improving resolution. The high-metaphase method prolongs the treatment time of mitotic arrest agents, allowing as many cells as possible to remain in metaphase, thus obtaining a large number of mitotic phases. While both methods can obtain a large number of mitotic phases and high-resolution chromosome banding, they are complex, time-consuming, and require high-quality specimens and experimental conditions, making large-scale application in routine clinical laboratories difficult.

[0004] Therefore, this application is submitted. Summary of the Invention

[0005] This invention provides a highly adaptable and high-resolution method for preparing chromosome slides from bone marrow cells of malignant hematological diseases, ensuring a sufficient number of metaphase phases; optimized hypotonicity and two-step fixation ensure sufficient dispersion and complete preservation of chromosomes in each phase; low-position droplet ensures uniform distribution of chromosomes on the slide; and precise banding and staining parameters ensure that the bands of each chromosome are clearly distinguishable.

[0006] The present invention solves its technical problem by adopting the following technical solution: A method for preparing chromosome slides from bone marrow cells of malignant hematological diseases with high adaptability and high resolution includes the following steps: (1) Inoculate bone marrow cells into culture medium and culture for 41-47 h, add treatment agent, continue to culture for 1-4 h, centrifuge to obtain cell pellet 1; (2) Add pre-warmed potassium chloride solution to cell pellet 1, treat with water bath, then add 0.5~1.5mL fixative for pre-fixation, mix gently, centrifuge, discard supernatant to obtain cell pellet 2; (3) Add 6-10 mL of fixative to cell pellet 2, mix well, let stand for 30 minutes or more, then centrifuge, discard the supernatant, add 6-10 mL of fixative again, mix well, centrifuge, and obtain cell pellet 3. (4) Add a small amount of fixative to cell pellet 3 to make the solution milky, and obtain cell suspension. Drop the cell suspension onto an ice slide, let it stand for about 1 minute, and then put it in the oven. (5) Baked slices; (6) Staining plate.

[0007] As an embodiment of this application, the culture medium is RPMI-1640 medium.

[0008] As an embodiment of this application, the amount of bone marrow cells inoculated is 1~5×10⁻⁶. 6 / mL.

[0009] As an embodiment of this application, the molar concentration of the potassium chloride solution is 0.05~0.1mol / L, and the amount of potassium chloride solution added is 5~10mL.

[0010] As an embodiment of this application, the fixative comprises methanol and glacial acetic acid in a volume ratio of (2~5):1.

[0011] As an embodiment of this application, the baking temperature of the baking sheet is 60~90℃ and the baking time is 1~4h.

[0012] As an implementation scheme of this application, the dyed slide is specifically prepared by: placing the glass slide in a first dyeing vat for digestion, then placing it in a second dyeing vat for rinsing, then placing it in a third dyeing vat for dyeing, removing it, then placing it in a fourth dyeing vat for rinsing, removing it, rinsing it clean with running water, and air-drying it naturally or by wind-drying.

[0013] As an embodiment of this application, the first dyeing vat contains 44-48 mL of physiological saline and 1.5-3.5 mL of trypsin solution, and the pH is adjusted to 7-7.2 with Tris-Base solution.

[0014] As an embodiment of this application, the second dyeing vat contains 40-50 mL of physiological saline.

[0015] As an embodiment of this application, the third staining vat contains 40-46 mL of double-distilled water, 4-6 mL of PBS buffer and 3-5 mL of Giemsa staining solution, with a pH of 7-7.2.

[0016] The beneficial effects of the present invention are as follows: (1) The present invention adopts a dual-concentration colchicine treatment, potassium chloride solution hypotonic treatment, two-step fixation treatment, low-position drop slide and precise banding staining scheme, which realizes a comprehensive improvement in the quality of chromosome specimens. It ensures a sufficient number of metaphase phases; the optimized hypotonic treatment and two-step fixation ensure that the chromosomes in each phase are fully dispersed and completely preserved; the low-position drop slide ensures that the chromosomes are evenly distributed on the slide; the precise banding staining parameters ensure that the bands of each chromosome are clearly distinguishable.

[0017] (2) The method of the present invention is applicable to bone marrow specimens of various hematological diseases such as acute non-lymphocytic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, and myelodysplastic syndrome. It has good specimen adaptability and clinical applicability. Attached Figure Description

[0018] Figure 1 This is a diagram showing the results of the experimental group.

[0019] Figure 2 This is a graph showing the effect of the control group.

[0020] Figure 3 This is a diagram of a combined dyeing vat.

[0021] Figures 4-8 This is the nucleation diagram of the experimental group.

[0022] Figures 9-13 This is the karyotype diagram for the control group. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0025] In this invention, numerical ranges are involved. Unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe features or characteristics, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0026] In this invention, there are no particular limitations on the specific dispersion and stirring methods.

[0027] Unless otherwise specified, all reagents or instruments used in this invention are commercially available conventional products. Unless otherwise specified, the raw materials used in each comparative example and the parallel experiments of each embodiment are the same commercially available products.

[0028] This application provides a method for preparing bone marrow cell chromosome slides, including the following steps: (1) Inoculate bone marrow cells into culture medium and culture for 41-47 h, add treatment agent, continue to culture for 1-4 h, centrifuge to obtain cell pellet 1; (2) Add potassium chloride solution to cell pellet 1, treat in a water bath (temperature 35~40℃, time 20~30min), add 0.5~1.5mL fixative, treat in a water bath (temperature 35~40℃, time 4~10min), centrifuge to obtain cell pellet 2; (3) Add 6-10 mL of fixative to cell pellet 2, mix well, centrifuge, discard the supernatant, add 5-8 mL of fixative, mix well, centrifuge, and obtain cell pellet 3; (4) Add fixative to cell precipitate 2 to make the solution milky, and obtain cell suspension. Drop the cell suspension onto an ice slide, let it stand for about 1 minute, and then put it in the oven. (5) Baked slices; (6) Staining plate.

[0029] This invention employs a two-step fixation method after hypotonic treatment with potassium chloride solution, consisting of pre-fixation and fixation. Specifically, after hypotonic treatment, 0.5-1.5 ml of fixative is added for pre-fixation, allowing the cell membrane to slowly permeate and the chromosomes to gradually shrink; then, 6-10 ml of fixative is added for full fixation. The pre-fixation step avoids cell rupture and chromosome loss caused by drastic changes in osmotic pressure when cells are suddenly exposed to a large amount of fixative. Simultaneously, the warm bath conditions during pre-fixation promote the stability and dispersion of chromosome structure. This approach ensures the integrity of cell morphology, promotes full chromosome dispersion, significantly reduces chromosome loss rate, and greatly improves the analyzability of mitotic phases.

[0030] In some embodiments, the culture temperature in step (1) is 36.5~37.5℃. In some embodiments, the bone marrow cells are derived from the iliac bone or sternum, the sample volume is 2-6 mL, stored at 2-8°C, and sent for testing as soon as possible within 6-8 hours.

[0031] In some embodiments, before inoculation, the specimen is smeared with Wright's staining solution to observe the proliferation of nucleated cells in the bone marrow, and the inoculation dosage is determined accordingly. For those with extremely low proliferation, the inoculation is about 0.5 ml; for those with low proliferation, the inoculation is 0.35-0.4 ml; for those with normal proliferation, the inoculation is 0.25-0.3 ml; for those with active proliferation, the inoculation is 0.2-0.25 ml; for those with relatively active proliferation, the inoculation is 0.15-0.2 ml; and for those with extremely active proliferation, the inoculation is 0.1 ml or inoculated drop by drop.

[0032] When aspirating bone marrow fluid, gradually increase the negative pressure; otherwise, the fluid will become excessively diluted. The sample volume must be greater than 2 ml, and it should be mixed immediately after collection, handled gently. When submitting the sample, include the patient's clinical diagnosis and peripheral blood count. Severe hemolysis, coagulation, or insufficient nucleated cells in the sample will directly affect bone marrow cell culture. Preserving the collected bone marrow in bone marrow culture medium is beneficial for bone marrow cell culture after submission.

[0033] In some embodiments, the culture medium is RPMI-1640 medium.

[0034] In some embodiments, the amount of bone marrow cells inoculated is 1 to 5 × 10⁶ / mL.

[0035] In some implementations, peripheral blood can be used instead of bone marrow for short-term culture in some hematologic malignancy patients. Prerequisites for peripheral blood collection from hematologic malignancy patients: 1) In cases where bone marrow aspiration is unavailable or no bone marrow can be obtained, the percentage of immature cells in the peripheral blood must be >20% for testing. 2) Peripheral blood can be collected for testing when the peripheral blood WBC count is >10 x 10^9 / L and the ratio of primitive to immature cells is >0.10. 3) Peripheral blood can be collected for testing in patients with CLL. 4) Somatic cell chromosomal abnormality detection is required.

[0036] When a patient's peripheral blood cell count is greater than 10 x 10^9 / L, and the percentage of cytokines and immature cells is greater than 10%, peripheral blood can be used instead of bone marrow for short-term culture. Patients undergoing chemotherapy or radiotherapy must wait at least one week after stopping treatment before undergoing bone marrow chromosome testing. Bone marrow collection is contraindicated in patients with coagulation disorders, local skin infections, tumors, or other conditions.

[0037] In some embodiments, the treatment agent is specifically added as follows: 50-200 μl of colchicine solution (i.e., colchicine stock solution) and 50-200 μl of colchicine solution diluted by half (i.e., colchicine stock solution diluted by half).

[0038] The concentration of the colchicine solution is 5~20 μg / ml.

[0039] This invention is the first to treat cell populations before the end of culture using one drop of stock colchicine and a 1-fold diluted colchicine solution. Cell populations with high proliferative activity are more sensitive to colchicine, and this concentration effectively arrests them at metaphase; while cell populations with low proliferative activity are less sensitive to colchicine, and the diluted colchicine solution is more conducive to their accumulation of mitotic phase. These two concentrations can accommodate cell populations with different proliferative activities in bone marrow specimens, maximizing the acquisition of metaphase.

[0040] In some embodiments, the molar concentration of the potassium chloride solution is 0.05~0.1 mol / L, and the amount of potassium chloride solution added is 5~10 mL.

[0041] In some embodiments, the fixative comprises methanol and glacial acetic acid in a volume ratio of (2~5):1.

[0042] In some embodiments, the baking temperature of the baking sheet is 60~90°C, and the baking time is 1~4 hours.

[0043] In some embodiments, the dyeing process specifically involves: placing the glass slide in a first dyeing vat for digestion, then rinsing it in a second dyeing vat, then dyeing it in a third dyeing vat, removing it, rinsing it in a fourth dyeing vat, removing it, rinsing it clean with running water, and then air-drying it naturally or by air drying.

[0044] In some embodiments, the first dyeing vat contains 44-48 mL of physiological saline and 1.5-3.5 mL of trypsin, and the pH is adjusted to 7-7.2 with Tris-Base solution.

[0045] In some embodiments, the second dye vat contains 40-50 mL of physiological saline.

[0046] In some embodiments, the third staining vat contains 40-46 mL of double-distilled water, 4-6 mL of PBS buffer, 3-5 mL of Giemsa staining solution, and has a pH of 7-7.2.

[0047] In some embodiments, the fourth dye vat contains 45-50 mL of tap water with a pH of 7-7.4. Rinse, remove, rinse thoroughly with running water, and air dry or air dry.

[0048] The specific steps for staining the slides are as follows: Add 44-48 mL of physiological saline and 1.5-3.5 mL of trypsin to the first staining tank, and adjust the pH to 7-7.2 with Tris-Base solution; add 40-50 mL of physiological saline to the second staining tank; add 40-46 mL of double-distilled water, 4-6 mL of PBS buffer, and 1-4 mL of Giemsa staining solution to the third staining tank; rinse with tap water in the fourth staining tank, remove the slides, rinse them thoroughly with running water, and air dry or air dry.

[0049] Place the glass slide in the first dyeing vat for digestion for 0.5 to 2 minutes, then place it in the second dyeing vat for rinsing, then place it in the third dyeing vat for dyeing for 3 to 5 minutes. Remove it, then place it in the fourth dyeing vat for rinsing, remove it, rinse it with running water, and let it air dry or air dry.

[0050] This invention provides precise control over key parameters of banding staining. Precise pH control of the staining vat ensures the stability of trypsin activity and Giemsa dye staining efficiency. Optimized trypsin concentration and digestion time ensure moderate hydrolysis of chromosome skeletal proteins, avoiding over-digestion that could lead to chromosome morphological damage. Appropriate Giemsa staining time and concentration result in moderate chromosome banding depth and clear contrast. This improves the reliability and comparability of laboratory test results.

[0051] In some embodiments, the centrifugation speed is 1200~1800 rpm and the centrifugation time is 8~20 min.

[0052] The present application is further illustrated below with specific embodiments:

[0053] Example 1 A method for preparing chromosome slides from bone marrow cells of malignant hematological diseases with high adaptability and high resolution includes the following steps: 1. Experimental Procedure 1.1 Specimen Collection 1.1.1. Collection requirements: Bone marrow is extracted from the iliac bone and sternum.

[0054] 1.1.2. Specimen type: heparin sodium anticoagulated blood or culture medium preservation.

[0055] 1.1.3. Specimen volume: 5±1ml.

[0056] 1.1.4. Storage container: disposable sterile heparin sodium anticoagulant tube or bone marrow culture medium.

[0057] 1.1.5. Specimen preservation: Store at 2–8℃ and send for testing as soon as possible within 6–8 hours; 1.1.6. Specimen Rejection Criteria: (1) The specimen lacks a unique barcode for identification; (2) Insufficient sample volume for testing; (3) The specimen was severely hemolyzed, coagulated, lipemic, or contaminated; (4) Specimens were anticoagulated using non-heparin sodium; (5) The number of nucleated cells in the specimen is too low.

[0058] 1.2. Reagents and Instruments 1.2.1. Reagents (1) Reagent composition: RPMI-1640 medium (bone marrow), colchicine, methanol, glacial acetic acid, trypsin, physiological saline, KCl, Tris-Base, Giemsa powder, glycerol, cedar oil, anhydrous ethanol, triple-distilled water, etc.

[0059] 1.2.2. Storage and Stability (1) Storage: RPMI-1640 medium should be stored at -20℃, colchicine and trypsin should be stored at 2-8℃, and other reagents should be stored at room temperature. Do not invert the container.

[0060] (2) Stability: When unopened and properly stored, it can remain stable until the indicated shelf life; when opened, it can remain stable for 12 weeks when properly stored.

[0061] 1.2.3. Usage Method (1) 1640 culture medium, colchicine, physiological saline, and cedar oil were used directly according to the instructions; (2) Trypsin, Giemsa powder, Tris-Base, etc. are prepared in advance as storage solutions, and can be diluted according to the ratio when used.

[0062] (3) Methanol, glacial acetic acid, KCl, anhydrous ethanol, etc. can be prepared into working solutions when used, and the fixative solution must be prepared fresh for use.

[0063] 1.2.4. Instruments CO2 incubator, clean bench, water bath, oven, horizontal centrifuge, microscope, automated chromosome analysis system, refrigerator, balance, etc.

[0064] 1.3. Quality Control Procedures 1.3.1. For each batch of newly arrived culture medium, and for each batch number of culture medium, a preliminary experiment must be conducted before use, and records must be kept. 1.3.2. Each batch of newly arrived methanol and glacial acetic acid must be tested before use, and records must be kept. 1.3.3 For each new batch of colchicine, a preliminary experiment must be conducted before use, and records must be kept. 1.3.4. For each batch of newly arrived Giemsa powder and trypsin, several batches of stock solution must be prepared in advance, and preliminary experiments must be conducted on each batch before use, with records kept.

[0065] 2. Operating Procedures (1) Cell culture: Nucleated cells were counted in the collected bone marrow samples. 0.2% glacial acetic acid was used as a diluent. The nucleated cell concentration of the submitted specimens was calculated under low magnification and the concentration was calculated according to 1~2×10⁻⁶ cells / mL. 6 Inoculate the culture flask with a volume of 1 ml / ml into RPMI-1640 medium; after inoculation, gently shake the flask horizontally several times to mix thoroughly, and incubate at 37°C for 48 hours (temperature difference ±0.5°C). Inoculation should be performed immediately after specimen collection.

[0066] (2) One hour before terminating the culture, add 100 μl of 10 μg / ml colchicine solution and 100 μl of colchicine solution diluted 1:1 to the culture flask, mix gently, and continue incubation at 37±0.5℃ for 1 hour (hereinafter referred to as the experimental group). Figure 1 As shown.

[0067] This step includes a control group (hereinafter referred to as the control group) with a single concentration of colchicine solution. 200 μl of a 10 μg / ml colchicine solution was directly added. Figure 2 As shown.

[0068] This application employs two concentrations of colchicine solution for treatment, which effectively arrests metaphase and facilitates the accumulation of mitotic phase. The two concentrations cater to cell populations with varying proliferative activities in bone marrow specimens, maximizing the acquisition of metaphase.

[0069] (3) Pour the fully cultured culture into two centrifuge tubes, label them, centrifuge at 1500 rpm for 10 minutes, discard the supernatant, and keep the cell pellet.

[0070] (4) Add 8 ml of 0.075 mol / L KCl solution preheated to 37℃ to the centrifuge tube, repeatedly blow and disperse the cells with a dropper, and incubate in a 37℃ water bath for 30 minutes.

[0071] (5) Slowly add 0.6-1 ml of freshly prepared fixative (methanol and glacial acetic acid in a volume ratio of 3:1, the same below) to the hypotonic cell suspension, mix gently, then centrifuge at 1500 rpm for 10 minutes, discard the supernatant, and leave the cell pellet.

[0072] (6) Slowly add 8 ml of fixative along the tube wall to the above-mentioned precipitated cells, mix gently, let stand for 30 minutes, centrifuge at 1500 rpm for 10 minutes, discard the supernatant, add 6 ml of fixative, mix gently, cover the centrifuge tube, and store in a refrigerator at 2℃~8℃ for later use.

[0073] (7) Centrifuge the prepared cell suspension at 1500 rpm for 10 minutes, discard the supernatant, leave the cell precipitate, add the newly prepared fixative, prepare the suspension to whey color, mix well, drop the slide about 10 cm above the prepared clean ice slide, drop 2 drops on each slide, spread evenly, and then flame it twice with an alcohol lamp.

[0074] (8) Place the prepared glass slide in a 75°C oven for one hour or bake at 90°C for one hour.

[0075] (9) Take four combined staining jars and place them in a 37℃ water bath. Add 40-45ml of physiological saline, 1.5-3.5ml of trypsin, and about 8 drops of Tris-Base solution to stain jar I to adjust the pH to 7.0-7.2 for chromosome digestion and banding. Add about 50ml of physiological saline to stain jar II for rinsing. Add 40-45ml of double-distilled water, 3-5ml of PBS buffer, and 3-5ml of Giemsa staining solution to stain jar III, mix well, and adjust the pH to 7.0-7.2 for staining slides. Add tap water to stain jar IV for rinsing. Figure 3 As shown.

[0076] (10) Take the aged specimen slide and place it in staining tank I. Digest with trypsin and show banding for about 10-40 seconds. Quickly take it out and place it in staining tank II. Rinse it gently a few times. Then put it in staining tank III for 1-5 minutes. Take it out and put it in staining tank IV to rinse 2-3 times. Then take it under the tap and rinse it clean. Let it air dry or air dry (the experimental operation time depends on the specific situation and needs to be determined in advance for the test slide and pre-staining). The staining method of this application can effectively improve the staining effect, increase efficiency and reduce staining time.

[0077] (11) After the slide is completely dry, select well-dispersed and appropriately sized cleavage phases under a microscope at low magnification, and then analyze them under an oil immersion microscope. Count 20 karyotypes for each specimen, analyze at least 5 karyotypes, and draw 5 karyotypes.

[0078] The karyotype diagram of the experimental group is as follows: Figures 4-8 As shown; the karyotype of the control group is shown. Figures 9-13 As shown.

[0079] (12) Reference values: Normal male: 46, XY; Normal female: 46, XX.

[0080] from Figures 4-13 As can be seen, the method described in this application can effectively improve and ensure the uniform distribution of chromosomes on the slide, effectively increase the length and size of chromosomes, improve the clarity of banding, and ensure that the banding of each chromosome is clearly distinguishable.

[0081] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the technical spirit of the present invention. The technical scope of the present invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A method for preparing chromosome slides from bone marrow cells of malignant hematological diseases with high adaptability and high resolution, characterized in that, Includes the following steps: (1) Inoculate bone marrow cells into culture medium and culture for 41-47 h, add treatment agent, continue to culture for 1-4 h, centrifuge to obtain cell pellet 1; (2) Add pre-warmed potassium chloride solution to cell pellet 1, treat with water bath, then add 0.5~1.5mL fixative for pre-fixation, mix gently, centrifuge, discard supernatant to obtain cell pellet 2; (3) Add 6-10 mL of fixative to cell pellet 2, mix well, let stand for 30 minutes or more, then centrifuge, discard the supernatant, add 6-10 mL of fixative again, mix well, centrifuge, and obtain cell pellet 3. (4) Add a small amount of fixative to cell pellet 3 to make the solution milky, and obtain cell suspension. Drop the cell suspension onto an ice slide, let it stand for about 1 minute, and then put it in the oven. (5) Baked slices; (6) Digestion; (7) Staining.

2. The method for preparing high-fitting, high-resolution bone marrow cell chromosome slides for malignant hematological diseases according to claim 1, characterized in that, The culture medium is bone marrow RPMI-1640 medium.

3. The method for preparing high-fitting, high-resolution bone marrow cell chromosome slides for malignant hematological diseases according to claim 1, characterized in that, The inoculation dose of the bone marrow cells is 1~5×10⁻⁶. 6 / mL.

4. The method for preparing high-fitting, high-resolution bone marrow cell chromosome slides for malignant hematological diseases according to claim 1, characterized in that, The molar concentration of the potassium chloride solution is 0.05~0.1mol / L, and the amount of potassium chloride solution added is 5~10mL.

5. The method for preparing high-fitting, high-resolution bone marrow cell chromosome slides for malignant hematological diseases according to claim 1, characterized in that, The fixative comprises methanol and glacial acetic acid in a volume ratio of (2~5):

1.

6. The method for preparing high-fitting, high-resolution bone marrow cell chromosome slides for malignant hematological diseases according to claim 1, characterized in that, The baking temperature of the wafer is 60~90℃, and the baking time is 1~4h.

7. The method for preparing high-fitting, high-resolution bone marrow cell chromosome slides for malignant hematological diseases according to claim 1, characterized in that, The specific process for dyeing the slides is as follows: the slides are placed in the first dyeing vat for digestion, then placed in the second dyeing vat for rinsing, then placed in the third dyeing vat for dyeing, removed, then placed in the fourth dyeing vat for rinsing, removed, rinsed clean with running water, and then air-dried or air-dried.

8. The method for preparing high-fitting, high-resolution bone marrow cell chromosome slides for malignant hematological diseases according to claim 7, characterized in that, The first dyeing vat contains 44-48 mL of physiological saline and 1.5-3.5 mL of trypsin solution, and the pH is adjusted to 7-7.2 with Tris-Base solution.

9. The method for preparing high-fitting, high-resolution bone marrow cell chromosome slides for malignant hematological diseases according to claim 7, characterized in that, The second dyeing vat contains 40-50 mL of physiological saline.

10. The method for preparing high-fitting, high-resolution bone marrow cell chromosome slides for malignant hematological diseases according to claim 7, characterized in that, The third staining tank contains 40-46 mL of double-distilled water, 4-6 mL of PBS buffer, and 3-5 mL of Giemsa staining solution, with a pH of 7-7.

2. The fourth dyeing vat contains 45-50 mL of tap water with a pH of 7-7.4.

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