Preparation method of leukemia bone marrow chromosome specimen

Abstract

The invention discloses a preparation method of a leukemia bone marrow chromosome specimen, and belongs to the field of biomedicine. According to the preparation method, the chromosome diffusion effect can be effectively improved, and the chromosome morphology is optimized; the repeatability is high, and the personnel operation error is low; and the safety is good, and no open fire is needed. Meanwhile, the success rate and the anomaly detection rate of bone marrow chromosomes are greatly improved. Therefore, the preparation method disclosed by the invention has important significance on leukemia bone marrow chromosome preparation and technical universality, and can effectively improve the value of cytogenetics in the aspects of diagnosis, treatment and prognosis of leukemia.

Description

Technical Field

[0001] The invention relates to the field of biomedical technology, and in particular to a method for preparing a leukemia bone marrow chromosome specimen. Background Art

[0002] Leukemia is a malignant clonal disease originating from hematopoietic stem cells. Its complex pathogenesis is associated with multiple factors such as biology, physics, chemistry, genetics and other blood diseases. Among them, the proliferation of leukemia cells in the bone marrow and other hematopoietic tissues is out of control, resulting in the inhibition of normal hematopoiesis and infiltration into other organs. It is reported that the number of leukemia cases in 2022 ranks among the top 15 cancers. Accurate diagnosis and typing of leukemia is the prerequisite for the correct selection of chemotherapy and treatment plans. At present, the common typing at home and abroad is cell morphology (Morphology), immunology (Immunology), cytogenetics (Cytogenetics) and molecular biology (Molecular biology), namely MICM typing. Bone marrow chromosome karyotype analysis is the main analytical method of cytogenetics, and plays an important role in the diagnosis, treatment and prognosis of leukemia. According to statistics, about 80% to 85% of leukemia patients have abnormal chromosome karyotypes. In 2001, the World Health Organization (WHO) revised the classification rules for lymphohematopoietic system diseases and used chromosome karyotype analysis as the basis for classification, which made the role of chromosome karyotype analysis more important.

[0003] At present, bone marrow chromosome preparation is divided into two technical processes: culture and preparation. The culture technology of chromosome preparation is divided into four methods: direct method, short-term culture method, synchronization method and immediate hypotonic method. The obtained cell suspension is used for preparation. Preparation mainly includes two drop slice methods: wet ice slice without fire method and wet ice slice over fire method.

[0004] However, the direct method has the problem of short chromosomes, bifurcated hairs and low success rate in the above-mentioned culture methods; the immediate hypotonic method has the problem of low abnormal detection rate; the short-term culture method requires aseptic operation, and the success rate and abnormal detection rate are low for certain types of leukemia; the synchronization method has high requirements for operation technology and low division index. Among the preparation methods, the wet ice tablet method without over-firing has the problem of poor chromosome dispersion and deep cytoplasm; the wet ice tablet over-firing method has the problem of short and thick chromosomes, poor dispersion and poor safety. In order to improve the above problems, the present invention is specially proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing leukemia bone marrow chromosome specimens. The preparation method of the present invention can safely and effectively improve the chromosome spreading effect and optimize the chromosome morphology, greatly improving the success rate and abnormality detection rate of bone marrow chromosomes.

[0006] The present invention is achieved in that:

[0007] The present invention provides a method for preparing a leukemia bone marrow chromosome specimen, comprising culturing bone marrow cells. The bone marrow cell culture comprises:

[0008] Inoculating part of the bone marrow cells in a culture medium for cultivation, adding a treatment agent to stop their division, and then adding a fixative for pre-fixation to obtain first precipitated cells;

[0009] Another part of bone marrow cells is added to a hypotonic solution, and a treatment agent is added to stop the division of the cells. After the hypotonic treatment, a fixative is added for pre-fixation to obtain a second precipitated cell.

[0010] The first precipitated cells and the second precipitated cells are mixed, and then a fixative is added for fixation to obtain a cell suspension.

[0011] In some embodiments, when preparing the first precipitated cells, the cell concentration of bone marrow cells in the culture medium is 1×10 6 / mL~2×10 6 / mL, the culture time is 24h-72h; the treatment agent is added to 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 preparing the second precipitated cells, the bone marrow cells are added to a hypotonic solution preheated at 37°C, 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. After adding the treatment agent, it is placed in a hypotonic environment at 37℃ for 30-40min.

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

[0014] The treatment agent may also be colchicine. Those skilled in the art may adjust the type and concentration according to actual needs, and the present invention does not limit this.

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

[0016] In some embodiments, the preparation method further comprises tableting, which comprises:

[0017] Add pre-cooled pure water to the pre-cooled slide to form a continuous water film with a certain thickness. Then pinch the frosted end of the slide to keep it horizontal, and add cell suspension 8-10 cm above the slide. Place the slide 1-5 cm above the infrared lamp, and then air-dry and bake the slide to complete the slide preparation.

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

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

[0020] In some embodiments, the slide is exposed to the infrared lamp for 3-7 seconds.

[0021] In some embodiments, the temperature of the baking sheet is 75-80° C. and the time is 2-3 hours.

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

[0023] The present invention improves the existing bone marrow cell culture method and preparation method to obtain a new method for preparing leukemia bone marrow chromosome specimens. The method has the advantages of the existing methods and can also improve their defects. It can not only effectively improve the chromosome diffusion effect and optimize the chromosome morphology; it has strong repeatability and low operator operation error; it is also safe and does not require open flames. At the same time, the success rate and abnormality detection rate of bone marrow chromosomes are greatly improved. Therefore, the preparation method of the present invention is of great significance for the preparation of leukemia bone marrow chromosomes and the popularization of technology, and can effectively improve the value of cytogenetics in the diagnosis, treatment and prognosis of leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 This is a comparison chart of tableting effects under different amounts of pre-cooled pure water treatment;

[0026] Figure 2 This is a comparison chart of the effects of different film-making methods. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0028] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0029] Materials and reagents involved in the following experiments

[0030] Reagents: bone marrow cell culture medium (purchased from Shanghai Lechen Biotechnology Co., Ltd., product number CM-M-50), trypsin (purchased from GIBCO), Giemsa staining solution (purchased from Heneng Biotechnology), colcemid (purchased from Lechen Biotechnology).

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

[0032] Example 1

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

[0034] 1) Leukemia patients in our hospital were randomly divided into five groups, A, E, and C according to the order of their visits. Group A was prepared using the modified method for preparing bone marrow chromosomes in this study; Group B was prepared using the direct method; Group C was prepared using the short-term culture method; Group D was prepared using the immediate hypotonic method; and Group E was prepared using the synchronization method. Each group had approximately 50 patients, with no fewer than 10 patients in Group E.

[0035] 2) Heparin anticoagulated bone marrow at a cell count of 1.5×10 6 / mL was inoculated in 5mL bone marrow cell culture medium and cultured for 24h. Colchicine (final concentration was 0.2μg / mL) was added 2h before harvesting. For A1 tube, 1mL of methanol glacial acetic acid solution (v / v=3:1) was added for pre-fixation;

[0036] 3) Take another 6×10 6 Bone marrow cells were preheated to 8 mL of 0.075 mol / L KCl solution at 37°C, and colchicine (final concentration 0.1 μg / mL) was added, and placed in a 37°C water bath for hypotonicity for 40 min. For tube A2, 1 mL of methanol-glacial acetic acid solution (v / v = 3:1) was added for pre-fixation;

[0037] 4) After centrifugation, the precipitated cells in tubes A1 and A2 were combined into tube A and fixed three times with methanol-glacial acetic acid solution (v / v=3:1);

[0038] 5) The preparation method uses the semi-wet ice sheet infrared lamp method: put the wash-free slide in a 4℃ refrigerator, pre-cool for 2 hours, take it out, add 0.5mL of 4℃ pre-cooled pure water to form a continuous water film with a certain thickness, hold the frosted end of the slide with your left hand, keep it horizontal, and add 30μL of cell suspension with your right hand at a height of 10cm from the slide. Place it about 3cm above the infrared lamp for 5s, remove the slide, air dry it, and bake it on a 75℃ baking machine for 3h;

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

[0040] Comparative Example 1

[0041] Direct method: Take 1.5×10 heparin anticoagulated bone marrow 6 / mL 0.5mL was inoculated in 10mL RPM1640 culture medium containing 20% ​​calf serum and colchicine (final concentration 0.2μg / mL), cultured in a 37℃ incubator for 4h, treated with 8mL 0.075mol / L KCl solution for 30min, and fixed with methanol glacial acetic acid solution (v / v=3:1) for 3 times.

[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 hypotonicity 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 for treatment, the cells were collected, and the hypotonic fixation treatment was the same as that of 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 40 min of hypotonicity. Cells were collected and fixed in the same manner as in Comparative Example 1.

[0048] Comparative Example 5

[0049] That is, the wet ice slide method: put the wash-free slide in 4℃ ice water, pre-cool for 2 hours, take it out, hold the frosted end of the slide with your left hand, keep it horizontal, add 30μL of cell suspension at a height of 3cm from the slide with your right hand, wait for the cell suspension to diffuse for a few seconds, and bake the slide in a 75℃ oven for 3 hours. After trypsin digestion, Giemsa staining.

[0050] Comparative Example 6

[0051] That is, the wet ice slide fire method: put the wash-free slide in 4℃ ice water, precool for 2 hours, take it out, hold the frosted end of the slide with your left hand, keep it horizontal, and add 30μL of cell suspension at a height of 3cm from the slide with your right hand. Wait for the cell suspension to spread for a few seconds, and use the inner flame of the alcohol lamp to burn it. When the slide is on fire, remove the slide, air dry it, and bake it on a 75℃ oven for 3 hours. After trypsin digestion, Giemsa staining.

[0052] Experimental Example 1

[0053] The effects of the chromosome specimens prepared in Example 1 and Comparative Examples 1-4 were compared. Successful chromosome preparation was defined as each slide having more than 5 cell division phases available for analysis. The criteria for judging abnormal karyotypes are described in accordance with the International System of Nomenclature for Human Cytogenetics (ISCN2020). At least 2 cells have the same chromosome gain or structural rearrangement, or 3 cells have the same chromosome loss to be confirmed as an abnormal clone. The chromosome preparation success rate and abnormality rate are 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. The chromosome preparation success rate of bone marrow cell chromosomes prepared by the improved method 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 Summary of the success rate and abnormality rate of four bone marrow chromosome culture methods

[0057]

[0058]

[0059] Experimental Example 2

[0060] 1. Comparison of tableting effects under different addition amounts of pre-cooled pure water treatment

[0061] Place the wash-free slide in a 4℃ refrigerator, precool for 2h, take it out, add 300μL (slide number A1), 0.5mL (slide number A2), 1mL (slide number A3), 3mL (slide number A4) of 4℃ precooled sterile water, respectively, to form a continuous water film with a certain thickness, hold the frosted end of the slide with your left hand, keep it horizontal, and add 30μL of cell suspension with your right hand at a height of 10cm from the slide. Place it about 5cm above the alcohol lamp / infrared lamp for 3-7s, remove the slide, air dry it, and bake it on a 75℃ baking machine for 3h.

[0062] Result analysis: A well-colored mitotic phase means that most of the chromosomes in the mitotic phase are stained moderately or do not hinder observation, and there should be no completely unstained chromosomes. The chromosome good coloring rate refers to the percentage of well-colored mitotic phases in the observed mitotic phases. A good mitotic phase means that the chromosomes in the mitotic phase are of moderate length and well dispersed, the number of chromosomes is 45±1, the number of overlapping chromosomes does not exceed two pairs and the overlapping part does not exceed 1 / 3 of the shorter chromosome, and the chromosomes are over-dispersed and should not exceed one oil immersion field of view. The good mitotic rate refers to the percentage of good mitotic phases in the observed mitotic phases.

[0063] like Figure 1 As shown, the semi-wet ice sheet infrared lamp method retains 0.5 mL of water film during preparation, and the chromosome coloring and division phase effects are optimal.

[0064] 2. The methods of Example 1 and Comparative Examples 5-6 were used to prepare tablets, which were numbered A, B and C respectively.

[0065] Evaluation of experimental results: Figure 2 As shown in Table 2, the semi-wet ice sheet infrared lamp method has a higher rate of good chromosome coloring and good mitotic phases than the wet ice sheet non-over-fire method and the wet ice sheet over-fire method; there is no obvious cytoplasm in the mitotic phase, each chromosome has an appropriate interval, most of the chromosomes do not overlap, the complete mitotic phases are all within one oil microscope field of view, and the degree of diffusion is good.

[0066] Table 2 Summary of chromosome coloring rate and mitotic phase rate

[0067] Production method Chromosome coloring rate (%) Good splitting rate (%) Wet ice flakes method 86.67 48.33 Wet ice flake fire method 81.67 76.67 Semi-wet ice flake infrared lamp method 91.67 90.00

[0068] Experimental Example 3 Evaluation of the Improved Method for Preparing Chromosomes from Leukemia Bone Marrow

[0069] Precision (repeatability): In the same experiment, the same experimenter prepared slices for the same sample according to the wet ice tablet without over-firing method, wet ice tablet over-firing method and semi-wet ice tablet infrared lamp method (i.e., Example 1), and the experiment was repeated at least 6 times to examine the repeatability of the method. Acceptance criteria: RSD of the test results of 6 repeated experiments for each method is ≤20%.

[0070] The experimental results are shown in Table 3. The same experimenter independently repeated the experiment 6 times and the test results showed that the RSD of the wet ice tablet without firing method 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 tablet without firing method (RSD=11.04%).

[0071] Table 3 Summary of the repeatability validation results for three bone marrow chromosome preparation methods

[0072]

[0073]

[0074] 2) Intermediate precision (inter-day variation): The same experimenter prepared the slices according to the wet ice tablet without over-firing method, wet ice tablet over-firing method and semi-wet ice tablet infrared lamp method (i.e., Example 1) on 3 different working days, and examined the inter-day variation of the methods. Acceptance criteria: RSD of the good splitting phase rate of the slices prepared by the preparation method at 3 different times is ≤20%.

[0075] The experimental results are shown in Table 4. The same experimenter tested the three preparation methods on three different working days. The RSD of the wet ice tablet burning method and the improved method was ≤20%, which met the verification requirements of the intermediate precision (inter-day difference); the improved method (RSD = 5.56%) was lower and more stable than the wet ice tablet burning method (RSD = 17.64%).

[0076] Table 4 Summary of intermediate precision (inter-day difference) verification results

[0077]

[0078] 3) Intermediate precision (personnel error): The same specimen was tested once by three experimenters for each preparation method to examine the influence of the method's personnel error. Acceptance criteria: RSD of the good splitting rate of each preparation method prepared by three experimenters was ≤20%.

[0079] The experimental results are shown in Table 5. The same test product was tested by three experimenters using three preparation methods. The RSD of the wet ice tablet unfired method and the improved method was ≤20%, which met the verification requirements of intermediate precision (personnel error). The improved method (RSD = 3.15%) was lower and more stable than the wet ice tablet overfired method (RSD = 5.09%).

[0080] Table 5 Summary of intermediate precision (personnel error) verification results

[0081]

[0082] According to the above-mentioned precision (repeatability), intermediate precision (inter-day difference) and intermediate precision (personnel error) test results, the leukemia bone marrow chromosome preparation method was evaluated. The semi-wet ice tablet infrared lamp method was compared with the wet ice tablet non-fired method and the wet ice tablet over-fired method. The RSDs of precision (repeatability), intermediate precision (inter-day difference) and intermediate precision (personnel error) were all ≤20%, and the preparation effect was more stable.

[0083] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a leukemia bone marrow chromosome specimen, characterized in that: The preparation method includes culturing bone marrow cells, and the bone marrow cell culture includes: Inoculating part of the bone marrow cells in a culture medium for cultivation, adding a treatment agent to stop their division, and then adding a fixative for pre-fixation to obtain first precipitated cells; Another part of bone marrow cells is added to a hypotonic solution, and a treatment agent is added to stop the division of the cells. After the hypotonic treatment, a fixative is added for pre-fixation to obtain a second precipitated cell. The first precipitated cells and the second precipitated cells are mixed, and then a fixative is added for fixation to obtain a cell suspension.

2. The preparation method according to claim 1, characterized in that: When preparing the first precipitated cells, the cell concentration of bone marrow cells in the culture medium is 1×10 6 / mL-2×10 6 / mL, the culture time is 24h-72h; the treatment agent is added to the culture medium 1-3h before the end of the culture, and the final concentration of the treatment agent is 0.2μg / mL.

3. The preparation method according to claim 1, characterized in that: When preparing the second precipitated cells, the bone marrow cells were added to a hypotonic solution preheated at 37°C, wherein the concentration of bone marrow cells in the hypotonic solution was 0.375×10 6 / mL-1.25×10 6 / mL; the final concentration of the treatment agent is 0.1 μg / mL. After adding the treatment agent, it is placed in a hypotonic environment at 37°C for 30-40 minutes.

4. The preparation method according to claim 1, characterized in that: The treatment agent includes colchicine and colchicine; and the hypotonic solution includes potassium chloride.

5. The preparation method according to claim 4, characterized in that: The fixative includes methanol glacial acetic acid solution (v / v=3:1), the ratio of the added volume of the fixative to the volume of the culture medium is 1:5, and the ratio of the concentration of the hypotonic solution to the added volume of the fixative is 0.6:1 (mol / mL).

6. The preparation method according to any one of claims 1 to 5, characterized in that: The preparation method further comprises tableting, which comprises: Add pre-cooled pure water to the pre-cooled slide to form a continuous water film with a certain thickness. Then pinch the frosted end of the slide to keep it horizontal, and add cell suspension 8-10 cm above the slide. Place the slide 1-5 cm above the infrared lamp, and then air-dry and bake the slide to complete the slide preparation.

7. The preparation method according to claim 6, characterized in that: The precooling temperature of the slide glass is 4°C, and the temperature of the precooling pure water is 4°C.

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

9. The preparation method according to claim 6, characterized in that: The slides were treated above the infrared lamp for 3-7 seconds.

10. The preparation method according to claim 6, characterized in that: The temperature of the baking sheet is 75-80° C. and the baking time is 2-3 hours.

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