A method for preparing a bone marrow chromosome specimen of leukemia

Abstract

The application discloses a preparation method of leukemia bone marrow chromosome specimens and belongs to the biomedical field. The preparation method can effectively improve chromosome diffusion effect and optimize chromosome morphology, has high repeatability, low personnel operation error, good safety and no need of open fire. Meanwhile, the success rate of bone marrow chromosome and the abnormal detection rate are greatly improved. Therefore, the preparation method has important significance for leukemia bone marrow chromosome preparation and technology popularization, and can effectively improve the value of cytogenetics in leukemia diagnosis, treatment and prognosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the biomedical technology field, in particular to a preparation method of leukemia bone marrow chromosome specimen. BACKGROUND

[0002] Leukemia is a malignant clonal disease derived from hematopoietic stem cells, and the complex pathogenesis is associated with multiple factors such as biology, physics, chemistry, genetics and other blood diseases. The uncontrolled proliferation of leukemia cells in bone marrow and other hematopoietic tissues leads to inhibition of normal hematopoiesis and infiltration into other organs. Accurate diagnosis of leukemia is the premise of correct selection of chemotherapy and treatment programs. Currently, the common method at home and abroad is MICM typing based on cell morphology (Morphology), immunology (Immunology), cytogenetics (Cytogenetics) and molecular biology (Molecular biology). Bone marrow chromosome karyotype analysis is the main analysis method of cytogenetics, and plays an important role in the diagnosis, treatment and prognosis of leukemia.

[0003] At present, the preparation of bone marrow chromosomes is divided into two technical processes of culture and preparation. The culture technology of chromosome preparation includes four methods: direct method, short-term culture method, synchronization method and immediate low permeation method. The obtained cell suspension is prepared into a sheet, and the preparation mainly includes two drop sheet methods: wet ice sheet without fire and wet ice sheet with fire.

[0004] However, the direct method in the above culture method has the problems of short and hairy chromosome, low success rate; the immediate low permeation method has the problem of low abnormal detection rate; the short-term culture method needs sterile operation, and has low success rate and abnormal detection rate for some types of leukemia; the synchronization method has high operation technical requirements and low division index. And the wet ice sheet without fire method has the problems of poor chromosome dispersion and deep cytoplasm; the wet ice sheet with fire method has the problems of short and thick chromosomes, poor dispersion degree and poor safety. In order to improve the above problems, the present application is proposed. SUMMARY

[0005] The purpose of the present application is to provide a preparation method of leukemia bone marrow chromosome specimen, which can safely and effectively improve the chromosome dispersion effect and optimize the chromosome morphology, greatly improving the success rate and abnormal detection rate of bone marrow chromosomes.

[0006] The present application is realized as follows:

[0007] The present application provides a preparation method of leukemia bone marrow chromosome specimen, including bone marrow cell culture. The bone marrow cell culture includes:

[0008] Part of the bone marrow cells are inoculated into a culture medium, and a treatment agent is added to stop the division of the cells, and then a fixing agent is added to pre-fix the cells to obtain first precipitated cells;

[0009] Another part of the bone marrow cells are added into a hypotonic solution, and a treatment agent is added to stop the division of the cells, and then a fixing agent is added to pre-fix the cells after hypotonic treatment to obtain second precipitated cells;

[0010] The first precipitated cells and the second precipitated cells are mixed, and then a fixing agent is added to fix the cells to obtain a cell suspension.

[0011] In some embodiments, when the first precipitated cells are prepared, the cell concentration of the bone marrow cells in the culture medium is 1×10 6 / mL~2×10 6 / mL, and the culture time is 24h-72h; the treatment agent is added into the culture medium 1-3h before the end of the culture, and the final concentration of the treatment agent is 0.2μg / mL.

[0012] In some embodiments, when the second precipitated cells are prepared, the bone marrow cells are added into a hypotonic solution preheated at 37℃, and the concentration of the bone marrow cells in the hypotonic solution is 0.375×10 6 / mL~1.25×10 6 / mL; the final concentration of the treatment agent is 0.1μg / mL, and the hypotonic treatment is performed at 37℃ for 30-40min after the treatment agent is added.

[0013] In some embodiments, the treatment agent includes colchicine; and the hypotonic solution includes potassium chloride.

[0014] The treatment agent can also be colchicine, and the type and concentration of the treatment agent can be adjusted according to actual needs, which are not limited in the present application.

[0015] In some embodiments, the fixing agent includes a methanol-ice acetic acid solution (v / v=3:1), and the ratio of the volume of the fixing agent to the volume of the culture medium is 1:5, and the ratio of the concentration of the hypotonic solution to the volume of the fixing agent is 0.6:1 (mol / mL).

[0016] In some embodiments, the preparation method further includes tablet preparation, which includes:

[0017] Pre-cooled pure water is added to a pre-cooled glass slide to form a continuous water film with a certain thickness, then the frosted end of the glass slide is pinched to keep it horizontal, and the cell suspension is added dropwise at a distance of 8-10cm above the glass slide, and the glass slide is placed at a distance of 1-5cm above an infrared lamp, and then the tablet is dried by air and baked to complete the tablet preparation.

[0018] In some embodiments, the pre-cooling temperature of the glass slide is 4℃, and the pre-cooling temperature of the pure water is 4℃.

[0019] In some embodiments, the dropwise amount of the cell suspension is 20-40 μL.

[0020] In some embodiments, the time for treating the slide above the infrared lamp is 3-7 s.

[0021] In some embodiments, the temperature for baking the slide is 75-80℃, and the time is 2-3 h.

[0022] The present application has the following beneficial effects:

[0023] The present application improves the existing bone marrow cell culture method and slide preparation method, and obtains a new preparation method of leukemia bone marrow chromosome specimen. The method has the advantages of the existing method and can improve the defects. The method can effectively improve the chromosome diffusion effect and optimize the chromosome morphology, has high repeatability and low personnel operation error, and has good safety without the participation of open fire. Meanwhile, the success rate of bone marrow chromosome and the abnormal detection rate are greatly improved. Therefore, the preparation method has important significance for the preparation and popularization of leukemia bone marrow chromosome technology, and can effectively improve the value of cytogenetics in the diagnosis, treatment and prognosis of leukemia. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0025] Figure 1 The preparation effect comparison chart under different pre-cooled pure water treatment amounts;

[0026] Figure 2 The effect comparison chart of different preparation methods. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions suggested by the manufacturer are used. If the production manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained by market purchase.

[0028] The features and properties of the present application will be further described in detail below in combination with the embodiments.

[0029] Materials and reagents involved in the following experiments

[0030] Reagents: bone marrow cell culture solution (purchased from Shanghai Ledan Biotech Co., Ltd., item number CM-M-50), trypsin (purchased from GIBCO), Giemsa staining solution (purchased from Hengneng Biotech), colchicine (purchased from Ledan Biotech).

[0031] Bone marrow cells: leukemia patients in Zhujiang Hospital of Southern Medical University.

[0032] Example 1

[0033] This example is a method for preparing a leukemia bone marrow chromosome specimen, and the steps are as follows:

[0034] 1) The leukemia patients in our hospital were randomly divided into five groups A-E according to the order of treatment. Group A was prepared by using the improved bone marrow chromosome preparation method of this study; group B was prepared by using the direct method; group C was prepared by using the short-term culture method; group D was prepared by using the immediate hypotonic method; and group E was prepared by using the synchronization method. Each group had about 50 patients, and group E had no less than 10 patients.

[0035] 2) Heparin-anticoagulated bone marrow was inoculated in 5 mL of bone marrow cell culture solution at a cell number of 1.5 x 10 6 / mL and cultured for 24 h. Two hours before harvesting, colchicine (final concentration 0.2 μg / mL) was added, and 1 mL of methanol glacial acetic acid solution (v / v = 3:1) was added for pre-fixation.

[0036] 3) Another 6 x 10 6 heparin-anticoagulated bone marrow cells were preheated in 8 mL of 0.075 mol / L KCl solution at 37°C, and colchicine (final concentration 0.1 μg / mL) was added. The mixture was placed in a 37°C water bath for 40 min for hypotonic treatment, and 1 mL of methanol glacial acetic acid solution (v / v = 3:1) was added for pre-fixation.

[0037] 4) After centrifugation, the cell pellets of A1 tube and A2 tube were combined into A tube, and methanol glacial acetic acid solution (v / v = 3:1) was added for fixation three times.

[0038] 5) The semi-wet ice slice infrared lamp method was used for slice preparation: the non-washed glass slide was pre-cooled in a 4°C refrigerator for 2 h, and then 0.5 mL of 4°C pre-cooled pure water was added to form a continuous water film with a certain thickness. The left hand held the frosted end of the glass slide horizontally, and the right hand was 10 cm away from the glass slide. 30 μL of cell suspension was added dropwise, and the glass slide was placed about 3 cm above the infrared lamp for 5 s. After moving away the glass slide, it was air-dried and then baked on a slice baking machine at 75°C for 3 h.

[0039] 6) After trypsin digestion, Giemsa staining was performed, and G-banding analysis was carried out under a microscope.

[0040] Comparative Example 1

[0041] The direct method: 1.5 x 106 / mL was inoculated with 0.5mL of culture medium containing 20% ​​calf serum RPM1640 and added with colchicine (final concentration 0.2μg / mL), cultured in a 37℃ incubator for 4h, treated hypotonic with 8mL of 0.075mol / L KCl solution for 30min, and fixed three times with methanol-glacial acetic acid solution (v / v = 3:1).

[0042] Comparative Example 2

[0043] Short-term culture method: heparin anticoagulated bone marrow cells at a cell count of 1.5×10 6 / mL was inoculated into 5mL culture medium and cultured for 24h. Colchicine was added 4h before harvest, and the cells were collected and fixed with hypotonic method in the same manner as in Comparative Example 1.

[0044] Comparative Example 3

[0045] Synchronization method: heparin anticoagulated bone marrow cells were counted at 1.5×10 6 / mL was inoculated into 5mL culture medium and cultured for 48h, 0.1μM 5-fluorodeoxyuridine and 4μM uridine were added and cultured for 17h, 10μM thymidine was added and cultured for another 6-8h, colchicine was added, the cells were collected, and hypotonic fixation treatment was the same as in Comparative Example 1.

[0046] Comparative Example 4

[0047] Immediate hypotonic method: take heparin anticoagulation 6×10 6 Bone marrow cells were directly inoculated into 8 mL of 0.075 mol / L KCl solution, and colchicine (final concentration 0.4 μg / mL) was added. The cells were placed in a 37° C. water bath for hypotonicity for 40 min, and the cells were collected and fixed as in Comparative Example 1.

[0048] Comparative Example 5

[0049] The wet slide method (not fire method) involves placing a pre-washed slide in 4°C ice water for 2 hours. Remove the slide and hold it horizontally with your left hand while holding the frosted end. Place 30 μL of the cell suspension 3 cm from the slide with your right hand. Allow the cell suspension to diffuse for a few seconds before baking the slide in a 75°C oven for 3 hours. After trypsinization, stain with Giemsa.

[0050] Comparative Example 6

[0051] Wet ice slide flame method: Place a wash-free slide in 4°C ice water for 2 hours. Remove the slide and hold it horizontally with your left hand while holding the frosted end. Place 30 μL of cell suspension 3 cm from the slide with your right hand. Allow the cell suspension to diffuse for a few seconds before using the inner flame of an alcohol lamp to flame the slide until it catches fire. Remove the slide, air dry, and bake it in a 75°C slide oven for 3 hours. After trypsinization, stain with Giemsa.

[0052] Experimental Example 1

[0053] The chromosome specimens prepared in Example 1 and Comparative Examples 1-4 were compared for effects, and the success of chromosome preparation was defined as having more than 5 analyzable cell division phases per slide. The abnormal karyotype judgment standard was described according to the International Nomenclature System for Human Cytogenetics (ISCN 2020). At least 2 cells had the same chromosome increase or structural rearrangement, or 3 cells had the same chromosome loss, which was confirmed as an abnormal clone. The chromosome preparation success rate and abnormality rate were calculated according to the following formula:

[0054]

[0055] Evaluation of experimental results: As shown in Table 1, the chromosome preparation success rate and chromosome abnormality rate were calculated according to the above formula, and the bone marrow cells were prepared for chromosome using the improved method, the chromosome preparation success rate was higher than that of the other four methods, and the chromosome abnormality rate was higher than that of the other four methods.

[0056] Table 1: Results of success rate and abnormality rate of four bone marrow chromosome culture methods

[0057]

[0058] Experimental Example 2

[0059] 1. Comparison of the effects of different amounts of pre-cooled pure water treatment

[0060] The non-washing glass slide was pre-cooled in a 4°C refrigerator for 2h, and then 300μL (slide No. A1), 0.5mL (slide No. A2), 1mL (slide No. A3), and 3mL (slide No. A4) of 4°C pre-cooled sterile water were added to form a continuous water film with a certain thickness. The left hand held the frosted end of the glass slide horizontally, and the right hand was 10cm away from the glass slide. 30μL of cell suspension was added. It was placed about 5cm above the alcohol lamp / infrared lamp, and moved away after 3-7s. After air drying, the slide was baked on a slide baking machine at 75°C for 3h.

[0061] Result analysis: well-colored division phase refers to most of the chromosomes in the division phase being moderately colored or not hindering observation, and there should be no completely uncolored chromosomes. The well-colored chromosome rate refers to the percentage of well-colored division phases in the observed division phases. Good division phase refers to the size and length of the chromosomes in the division phase being moderate, the dispersion being good, the number of chromosomes being 45±1, the overlapping chromosomes not exceeding two pairs and the overlapping part not exceeding 1 / 3 of the shorter chromosome, and the excessive dispersion of chromosomes not exceeding one oil lens field. The good division phase rate refers to the percentage of good division phases in the observed division phases.

[0062] As Figure 1As shown, the semi-wet ice sheet infrared lamp method retains 0.5 mL of water film during preparation, which has the best chromosome coloring and division phase effects.

[0063] 2. Tablets were prepared using the methods of Example 1 and Comparative Examples 5-6. The tablets were numbered A, B, and C, respectively.

[0064] Evaluation of experimental results: Figure 2 As shown in Table 2, the semi-wet ice slide infrared lamp method has a higher rate of good chromosome staining and good mitotic phases than the wet ice slide non-fired method and the wet ice slide overfired method; the mitotic phases have no obvious cytoplasm, each chromosome has an appropriate interval, most chromosomes do not overlap, and the complete mitotic phases are all within the field of view of an oil immersion lens, with a good degree of diffusion.

[0065] Table 2 Summary of chromosome coloring and mitotic phase results

[0066]

[0067] Experimental Example 3 Evaluation of an Improved Method for Preparing Chromosomes from Leukemia Bone Marrow

[0068] Precision (Repeatability): Within the same experiment, the same researcher prepared the same sample using the wet ice tablet method (unfired), wet ice tablet method (fired), and semi-wet ice tablet method (i.e., Example 1) at least six times to assess the reproducibility of the methods. Acceptance criterion: RSD ≤ 20% for the six replicates for each method.

[0069] The experimental results are shown in Table 3. The same experimenter independently repeated the experiment 6 times and tested the results. The RSD of the wet ice slice method without combustion and the improved method was ≤20%, which met the verification requirements of the method repeatability. The improved method (RSD=4.13%) was lower and more stable than the wet ice slice method without combustion (RSD=11.04%).

[0070] Table 3 Summary of the repeatability validation results for three bone marrow chromosome preparation methods

[0071]

[0072] 2) Intermediate precision (inter-day variability): The same experimenter prepared slabs using the wet ice tablet method (no ignition), the wet ice tablet method (ignition), and the semi-wet ice tablet method (i.e., Example 1) on three different working days to assess inter-day variability. Acceptance criterion: RSD of the good split phase rate for each method at the three different times was ≤20%.

[0073] The experimental results are shown in Table 4, and the same experimental personnel respectively on three different working days for the detection results of three kinds of preparation methods, wet ice piece over fire method and improved method RSD≤20%, meet the verification requirements of intermediate precision (inter-day difference); two improved methods (RSD=5.56%) are lower than wet ice piece over fire method (RSD=17.64%), and are more stable.

[0074] Table 4 Intermediate precision (inter-day difference) verification result summary table

[0075]

[0076] 3) Intermediate precision (personnel error): the same sample is independently tested by 3 experimental personnel for each preparation method 1 time, and the personnel error influence of the method is investigated. Acceptance standard: the RSD of the split phase good rate of each preparation method prepared by 3 experimental personnel is ≤20%.

[0077] The experimental results are shown in Table 5, and the same sample is tested by 3 experimental personnel for the detection results of three kinds of preparation methods, wet ice piece over fire method and improved method RSD≤20%, meet the verification requirements of intermediate precision (personnel error); two improved methods (RSD=3.15%) are lower than wet ice piece over fire method (RSD=5.09%), and are more stable.

[0078] Table 5 Intermediate precision (personnel error) verification result summary table

[0079]

[0080] According to the detection results of the above-mentioned precision (repeatability), intermediate precision (inter-day difference) and intermediate precision (personnel error), the leukemia bone marrow chromosome preparation method is evaluated, and the precision (repeatability), intermediate precision (inter-day difference) and intermediate precision (personnel error) RSD of semi-wet ice piece infrared lamp method compared with wet ice piece over fire method and wet ice piece over fire method are all ≤20%, and the preparation effect is more stable.

[0081] The above only describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a bone marrow chromosome specimen of leukemia, characterized by, The preparation method comprises bone marrow cell culture, and the bone marrow cell culture comprises: Part of the bone marrow cells are inoculated in a culture medium, and a treatment agent is added to stop the division of the bone marrow cells, then a fixing agent is added to pre-fix the bone marrow cells, and first precipitated cells are obtained; Another part of the bone marrow cells are added into a hypotonic solution, and a treatment agent is added to stop the division of the bone marrow cells, then a fixing agent is added to pre-fix the bone marrow cells after hypotonic treatment, and second precipitated cells are obtained; The first precipitated cells and the second precipitated cells are mixed, and a fixing agent is added to fix the cells, and a cell suspension is obtained.

2. The production method according to claim 1, characterized by, The cell concentration of bone marrow cells in the culture medium is 1 x 10 6 / mL to 2 x 10 6 / mL, and the culture time is 24 h to 72 h. The treating agent is added into the culture medium 1 h to 3 h before the end of the culture, and the final concentration of the treating agent is 0.2 μg / mL.

3. The preparation method according to claim 1, characterized in that For the preparation of the second population of precipitated cells, the bone marrow cells were added to a hypotonic solution pre-warmed at 37°C, at a concentration of 0.375 x 10 6 / mL - 1.25 x 10 6 / mL, and the final concentration of the treatment agent was 0.1 μg / mL. The cells were then incubated in the hypotonic solution at 37°C for 30-40 min.

4. The method of claim 1, wherein, The treatment agent comprises colchicine and colchicin, and the hypotonic solution comprises potassium chloride.

5. The production method according to claim 4, characterized by, The fixing agent comprises a methanol-ice acetic acid solution with a volume ratio of 3:1, the ratio of the adding volume of the fixing agent to the volume of the culture medium is 1:5, and the ratio of the concentration of the hypotonic solution to the adding volume of the fixing agent is 0.6:1 (mol / mL).

6. The method of any one of claims 1-5, wherein, The preparation method further comprises tabletting, and the tabletting comprises: Pre-cooled pure water is added to a pre-cooled glass slide to form a continuous water film with a certain thickness, then the frosted end of the glass slide is pinched to keep the glass slide horizontal, and the cell suspension is added dropwise at a distance of 8-10 cm above the glass slide, the glass slide is placed at a distance of 1-5 cm above an infrared lamp, and then the tabletting is completed after air drying and baking.

7. The preparation method according to claim 6, characterized in that The pre-cooling temperature of the glass slide is 4℃, and the temperature of the pre-cooled pure water is 4℃.

8. The preparation method according to claim 6, characterized in that The dropwise adding amount of the cell suspension is 20-40 μL.

9. The preparation method according to claim 6, characterized in that The treatment time of the glass slide above the infrared lamp is 3-7 s.

10. The method of claim 6, wherein, The baking temperature is 75-80℃, and the baking time is 2-3 h.

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