A somatic cell chromosome dispersion kit and dispersion method for chromosome karyotype analysis

Abstract

This invention discloses a somatic cell chromosome dispersion kit and method for chromosome karyotype analysis. The somatic cell chromosome dispersion method for chromosome karyotype analysis includes the following steps: S1, culturing a body fluid containing somatic cells in a cell culture medium; S2, adding acetaminophen solution to the cultured cell culture medium and incubating for 2-4 hours, wherein the final concentration of acetaminophen in the cell culture medium is 60 μg / mL-110 μg / mL; S3, adding colchicine to the incubated cell culture medium for 0.5-1.5 hours, wherein the final concentration of colchicine in the cell culture medium is 5 μg / mL-15 μg / mL. This somatic cell chromosome dispersion method can effectively improve the dispersion, banding clarity, and pass rate of chromosomes in somatic cells, and can effectively avoid the risk of chromosomal aberrations during the processing.

Description

Technical Field

[0001] This invention belongs to the field of karyotype analysis technology, specifically relating to a somatic cell chromosome dispersion kit and dispersion method for chromosome karyotype analysis. Background Technology

[0002] Chromosomal karyotype analysis is a core standard method for diagnosing chromosomal abnormality-related diseases. Its key lies in obtaining uniformly dispersed, morphologically intact, and clearly banded metaphase chromosomes. Current routine procedures primarily rely on spindle inhibitors such as colchicine to arrest cells in metaphase, followed by hypotonic treatment with 0.075 mol / L potassium chloride solution to induce cell swelling, and pre-fixation with Carnoy's fixative. Chromosomal dispersion and flattening are then achieved under specific temperature and humidity conditions. However, existing somatic cell chromosome dispersion methods have three significant drawbacks in terms of reagent systems and operational procedures: First, colchicine has a narrow concentration window; excessively high concentrations (e.g., greater than 10 μg / ml) can easily lead to excessive chromosome shrinkage, elongated morphology, and tangling, making effective dispersion difficult. This is especially problematic in fragile samples such as elderly individuals and cancer patients, where cell sensitivity to the drug varies greatly, resulting in significantly reduced compatibility. Secondly, colchicine alone provides insufficient clarity in chromosome banding, often requiring the use of auxiliary dispersants such as actinomycin D and mitomycin to improve banding resolution. However, these drugs themselves are genotoxic, and their combined use increases the risk of chromosome breakage, recombination, and other aberrations, affecting the accuracy of result interpretation. Thirdly, some samples that repeatedly fail karyotype analysis are highly sensitive to traditional reagents, and even after standard treatment, it is still difficult to obtain a sufficient quantity and quality of qualified metaphase cells, leading to detection failure or missed detection. Therefore, existing somatic cell chromosome dispersion methods still have significant shortcomings in terms of dispersion, banding clarity, pass rate, and sample compatibility. There is an urgent need to develop a dispersion method that can ensure uniform chromosome dispersion and clear banding, avoid introducing genotoxicity, and has good adaptability to diverse somatic cell samples. Summary of the Invention

[0003] The purpose of this invention is to provide a somatic cell chromosome dispersion method and kit for chromosome karyotype analysis, which can effectively improve the dispersion, morphological integrity and banding clarity of chromosomes in somatic cells.

[0004] The following technical solutions are used to achieve the above objectives.

[0005] The first aspect of this invention provides a method for somatic chromosome dispersion for chromosome karyotype analysis, comprising the following steps:

[0006] S1. Place the body fluid containing somatic cells in a cell culture medium for incubation;

[0007] S2. Add acetaminophen solution to the cell culture medium after culture is completed and incubate for 2h~4h. The final concentration of acetaminophen in the cell culture medium system is 60μg / mL~110μg / mL.

[0008] S3. Add colchicine to the incubated cell culture medium and treat for 0.5h~1.5h, wherein the final concentration of colchicine in the cell culture medium system is 5μg / mL~15μg / mL.

[0009] In some embodiments, step S2 is selected from at least one of the following:

[0010] The final concentration of acetaminophen in the cell culture medium system is 60 μg / mL to 100 μg / mL, preferably 90 μg / mL to 100 μg / mL, and more preferably 98 μg / mL to 100 μg / mL;

[0011] The incubation time is 2.5h to 3.5h, preferably 3.3h to 3.5h.

[0012] In some embodiments, step S3 is selected from at least one of the following:

[0013] The final concentration of colchicine in the cell culture medium system is 7 μg / mL to 13 μg / mL, preferably 9 μg / mL to 11 μg / mL, and more preferably 9.5 μg / mL to 10.5 μg / mL;

[0014] The processing time is 0.8h to 1.2h, preferably 0.9h to 1.1h.

[0015] In some embodiments, step S1, which involves culturing the body fluid containing somatic cells in a cell culture medium, specifically includes the following steps:

[0016] Somatic fluid containing somatic cells is inoculated into cell culture medium, and phytohemagglutinin is added to a final concentration of 15 μg / mL to 25 μg / mL. The mixture is then incubated at 36℃ to 38℃ in a 4% to 8% CO2 incubator for 65 to 80 hours, preferably 65 to 75 hours.

[0017] And / or, the body fluid containing somatic cells also contains an anticoagulant;

[0018] And / or, the body fluid is one of peripheral blood, amniotic fluid, bone marrow, and umbilical cord blood;

[0019] And / or, the volume ratio of the body fluid to the cell culture medium is 1:3~6, preferably 1:4.5~5.5.

[0020] In some implementations, the following steps are included after step S3:

[0021] Somatic cells treated with colchicine were collected and subjected to hypotonic treatment and fixation treatment in sequence.

[0022] In some embodiments, the hypotonic treatment involves treatment with a potassium chloride solution of concentration 0.01 mol / L to 0.1 mol / L for 25 min to 35 min;

[0023] And / or, the fixation treatment is performed using a fixative solution, wherein the fixative solution is a mixture of methanol and glacial acetic acid, and the volume ratio of methanol to glacial acetic acid is 2~4:1.

[0024] A second aspect of the present invention provides a somatic chromosome dispersion kit for chromosome karyotype analysis, comprising cell culture medium, acetaminophen, and colchicine; wherein the final concentration of acetaminophen in the cell culture medium is 60 μg / mL to 110 μg / mL, and the final concentration of colchicine in the cell culture medium is 5 μg / mL to 15 μg / mL.

[0025] In some embodiments, the final concentration of acetaminophen in the cell culture medium is 60 μg / mL to 100 μg / mL, preferably 90 μg / mL to 100 μg / mL, and more preferably 98 μg / mL to 100 μg / mL.

[0026] And / or, the final concentration of the colchicine in the cell culture medium is 7 μg / mL to 13 μg / mL, preferably 9 μg / mL to 11 μg / mL, and more preferably 9.5 μg / mL to 10.5 μg / mL.

[0027] In some embodiments, the cell culture medium consists of a basal culture medium supplemented with fetal bovine serum, L-glutamine, penicillin, and streptomycin;

[0028] The final concentration of the fetal bovine serum is 5%~15% v / v, the final concentration of L-glutamine is 1 mmol / L~10 mmol / L, the final concentration of penicillin is 80 U / mL~120 U / mL, and the final concentration of streptomycin is 80 μg / mL~120 μg / mL.

[0029] In some embodiments, the basal culture medium is RPMI 1640 medium;

[0030] And / or, the final concentration of the fetal bovine serum is 9%~11% v / v, the final concentration of the L-glutamine is 1 mmol / L~3 mmol / L, the final concentration of the penicillin is 95 U / mL~105 U / mL, and the final concentration of the streptomycin is 95 μg / mL~105 μg / mL.

[0031] In this invention, the inventors discovered through long-term cytogenetics research that using a specific concentration of acetaminophen in combination with colchicine, treating somatic cells in a specific order for a specific time, can effectively improve the dispersion, banding clarity, and pass rate of chromosomes within somatic cells. Furthermore, it can effectively avoid the risk of chromosomal aberrations during the treatment process. Compared to the traditional colchicine-only treatment system, chromosome dispersion can be improved by 15%-20%, the banding clarity pass rate can reach over 90%, and the pass rate is as high as 89%. This method is suitable for the dispersion processing of cellular chromosomes in body fluids such as peripheral blood, bone marrow, umbilical cord blood, and amniotic fluid, and is especially suitable for fragile samples such as peripheral blood from elderly patients or those with hematological malignancies, effectively solving the problem of repeated karyotype analysis failures. Attached Figure Description

[0032] Figure 1 This is the karyotype examined under an oil immersion microscope in Example 1. Figure 1 ;

[0033] Figure 2 This is the karyotype examined under an oil immersion microscope in Example 1. Figure 2 ;

[0034] Figure 3 This is the karyotype examined under an oil immersion microscope in Example 2. Figure 1 ;

[0035] Figure 4 This is the karyotype examined under an oil immersion microscope in Example 2. Figure 2 ;

[0036] Figure 5 This is the karyotype examined under an oil immersion microscope in Example 2. Figure 3 ;

[0037] Figure 6 This is the karyotype image obtained by oil immersion microscope examination in Example 3;

[0038] Figure 7 This is the karyotype image obtained by oil immersion microscopy in Example 4;

[0039] Figure 8 This is the karyotype image obtained by oil immersion microscope examination in Example 5;

[0040] Figure 9 This is the karyotype image obtained by oil immersion microscope examination in Example 6;

[0041] Figure 10 This is the karyotype image obtained by oil immersion microscope examination in Example 7;

[0042] Figure 11 This is the karyotype image obtained by oil immersion microscopy in Example 8;

[0043] Figure 12This is the karyotype image obtained by oil immersion microscopy in Example 9;

[0044] Figure 13 Nucleotype examination under an oil immersion microscope in Comparative Example 1 Figure 1 ;

[0045] Figure 14 Nucleotype examination under an oil immersion microscope in Comparative Example 1 Figure 2 . Detailed Implementation

[0046] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0047] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0048] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0049] The present invention will be further described in detail below with reference to specific embodiments.

[0050] Unless otherwise specified, all raw materials and reagents used in the following examples and comparative examples are commercially available products. Some of the raw materials are described below:

[0051] Acetaminophen (final concentration 80μg / mL-100μg / mL); CR Double-Crane Pharmaceutical Co., Ltd., specifications: 2ml-0.25g. National Drug Approval Number: H11020531.

[0052] Colchicine (final concentration 10 μg / mL); Sigma, catalog number: D7385-10mg.

[0053] Potassium chloride (concentration 0.075 mol / L): Guangdong Guanghua.

[0054] Cell culture medium: RPMI 1640 medium (containing 10% fetal bovine serum, 2 mmol / L L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin).

[0055] Supporting reagents: phytohemagglutinin (PHA, final concentration 20 μg / mL), hypotonic solution (0.075 mol / L KCl), fixative (methanol: glacial acetic acid = 3:1), 10% Giemsa staining solution (pH 6.8, diluted with PBS).

[0056] Example 1

[0057] This embodiment provides a somatic chromosome dispersion method for chromosome karyotype analysis, comprising the following steps:

[0058] 1) Experimental materials

[0059] Five peripheral blood samples from patients with hematologic malignancies (anticoagulated with sodium heparin, sourced from a sample bank approved by the ethics committee of a collaborating hospital); RPMI 1640 medium, PHA, acetaminophen (purity ≥99%), colchicine, potassium chloride hypotonic solution, Carnoy's fixative, Giemsa stain; 25cm 2 Culture flasks, pre-cooled glass slides, centrifuge, CO2 incubator, 100× oil immersion microscope.

[0060] 2) Implementation process

[0061] Step 1, Cell Seeding and Culture:

[0062] Heparin sodium anticoagulated peripheral blood samples (heparin sodium concentration 20 IU / ml (Yangpu Medical)) were inoculated into cell culture medium at a volume ratio of 1:5 (each sample was divided into 6 cell culture media for culture). PHA (phytohemagglutinin, final concentration 20 μg / mL) was added to the cell culture medium and cultured at 37℃ in a 5% CO2 incubator for 72 hours.

[0063] Step 2, Acetaminophen treatment:

[0064] After culturing the cells in the culture medium for 72 hours, add acetaminophen stock solution (100 mg / mL, dissolved in sterile water) to the cell culture medium to make the final concentration of acetaminophen in the cell culture medium 80 μg / mL, and continue incubation for 3.5 hours.

[0065] Step 3, colchicine treatment:

[0066] After incubation, colchicine was added to bring the final concentration of colchicine to 10 μg / mL, and the mixture was treated at 37°C for 1 hour.

[0067] Step 4, Hypotonicity and Fixation:

[0068] Collect cells treated with colchicine, centrifuge at 2300 r / min for 8 minutes, discard the supernatant, add 8 mL of preheated hypotonic buffer (potassium chloride) at 37℃, and treat with hypotonic buffer for 30 minutes; add 1 mL of fixative (methanol: glacial acetic acid = 3:1) for prefixation, centrifuge, discard the supernatant, add 5 mL of fixative and fix at room temperature for 30 minutes, repeat fixation twice.

[0069] Step 5, slide preparation and staining:

[0070] Take a pre-cooled glass slide, add 2-3 drops of cell suspension from a height of 15 cm, and allow it to air dry at room temperature; stain with 10% Giemsa solution for 15 minutes, rinse with running water, air dry, and examine under a microscope. The microscopic results are as follows: Figure 1 and Figure 2 As shown.

[0071] Example 2

[0072] This embodiment provides a somatic chromosome dispersion method for chromosome karyotype analysis, processed according to the method in Example 1. The difference from Example 1 is that the final concentration of acetaminophen is 100 μg / mL. All other addition amounts and preparation steps are the same. Microscopic examination results are as follows. Figures 3 to 5 As shown.

[0073] Example 3

[0074] This embodiment provides a somatic chromosome dispersion method for chromosome karyotype analysis, processed according to the method in Example 1. The difference from Example 1 is that the final concentration of acetaminophen is 60 μg / mL. All other addition amounts and preparation steps are the same. Microscopic examination results are as follows. Figure 6 As shown.

[0075] Example 4

[0076] This embodiment provides a somatic chromosome dispersion method for chromosome karyotype analysis, processed according to the method in Example 1. The difference from Example 1 is that the final concentration of acetaminophen is 120 μg / mL. All other addition amounts and preparation steps are the same. Microscopic examination results are as follows. Figure 7 As shown.

[0077] Example 5

[0078] This embodiment provides a method for somatic chromosome dispersion for chromosome karyotype analysis, processed according to the method in Example 1. The difference from Example 1 is that the final concentration of acetaminophen is 40 μg / mL. All other addition amounts and preparation steps are the same. Microscopic examination results are as follows. Figure 8 As shown.

[0079] Example 6

[0080] This embodiment provides a method for somatic chromosome dispersion for chromosome karyotype analysis, following the method in Example 1. The difference from Example 1 is that the acetaminophen incubation time is 1 hour. All other addition amounts and preparation steps are the same. Microscopic examination results are as follows. Figure 9 As shown.

[0081] Example 7

[0082] This embodiment provides a somatic chromosome dispersion method for chromosome karyotype analysis, processed according to the method in Example 1. The difference from Example 1 is that the acetaminophen incubation time is 2 hours. All other addition amounts and preparation steps are the same. Microscopic examination results are as follows. Figure 10 As shown.

[0083] Example 8

[0084] This embodiment provides a method for somatic chromosome dispersion for chromosome karyotype analysis, following the method in Example 1. The difference from Example 1 is that the acetaminophen incubation time is 2.5 hours. All other addition amounts and preparation steps are the same. Microscopic examination results are as follows. Figure 11 As shown.

[0085] Example 9

[0086] This embodiment provides a method for somatic chromosome dispersion for chromosome karyotype analysis, following the method in Example 1. The difference from Example 1 is that the acetaminophen incubation time is 5 hours. All other addition amounts and preparation steps are the same. Microscopic examination results are as follows. Figure 12 As shown.

[0087] Comparative Example 1

[0088] This comparative example provides a somatic chromosome dispersion method for chromosome karyotype analysis, processed according to the method of Example 1. The difference from Example 1 is that acetaminophen is not added. All other addition amounts and preparation steps are the same. Microscopic results are as follows. Figure 13 and Figure 14 As shown.

[0089] The slides prepared in the examples and comparative examples were subjected to microscopic analysis, and the specific methods are as follows:

[0090] Dispersion: According to the sample preparation of the example, the automatic scanning function of the Zeiss fully automatic microscope was used to automatically and randomly select 50 metaphase fields of view and record the total number of metaphases in all fields of view.

[0091] Acceptance criteria: According to the International Nomenclature System for Human Cell Genetics (ISCN) (2024), in qualified dispersed metaphase, chromosomes are not intertwined or overlapping, each chromosome has an independent outline, and the chromosome set is evenly distributed in the field of view without clustering (Zeiss microscopes can automatically identify chromosome edge outlines through image analysis software).

[0092] Dispersion calculation formula:

[0093]

[0094] Example: In Example 2, there were 200 effective split phases in 50 fields of view, and 176.6 qualified dispersed split phases, which is calculated to be 88.3%.

[0095] Calculation of bandgap resolution (%)

[0096] Judgment criteria: The qualified clarity of chromosome G banding in the cleavage phase is determined by Zeiss microscope image analysis. The bands are clear, the contrast between light and dark is obvious, the band spacing is uniform, the characteristic areas such as centromeres and telomeres are fully banded, and the gray value difference in Zeiss microscope image analysis software is ≥50.

[0097] Calculation formula

[0098]

[0099] Example: In Example 2, there were 187 qualified splitting phases showing banding, and a total of 200 effective splitting phases, which was calculated to be 93.5%.

[0100] Microscope model: Zeiss biological microscope, Axio imager Z2

[0101] The results are shown in Table 1 below. Figures 1 to 14 As shown.

[0102] Table 1

[0103]

[0104] From Table 1 and Figures 1-14The results showed that the combined use of acetaminophen and colchicine to treat peripheral blood cells effectively improved the dispersion, morphological integrity, and banding clarity of chromosomes in somatic cells. Examples 1-9 demonstrated that the concentration of acetaminophen and the incubation time mutually affected the staining effect. Higher concentrations of acetaminophen and shorter incubation times resulted in a decrease in the number of karyotypes, poor karyotype dispersion, and a significant decrease in dispersion and banding clarity, leading to a large number of untransformed cells. Therefore, controlling the acetaminophen concentration to 60 μg / mL-100 μg / mL and the incubation time to 2-4 hours effectively improved the dispersion, banding clarity, and pass rate of chromosomes in somatic cells. The optimal concentration of acetaminophen (100 μg / mL) and incubation time (3.5 hours) yielded the best dispersion, banding clarity, and pass rate.

[0105] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0106] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A somatic cell chromosome dispersion method for chromosome karyotyping, characterized by, Includes the following steps: S1. Place the body fluid containing somatic cells in a cell culture medium for incubation; S2. Add acetaminophen solution to the cell culture medium after culture is completed and incubate for 2h~4h. The final concentration of acetaminophen in the cell culture medium system is 60μg / mL~110μg / mL. S3. Add colchicine to the incubated cell culture medium and treat for 0.5h~1.5h, wherein the final concentration of colchicine in the cell culture medium system is 5μg / mL~15μg / mL.

2. The somatic cell chromosome dispersion method according to claim 1, wherein In step S2, at least one of the following methods is selected: The final concentration of acetaminophen in the cell culture medium system is 60 μg / mL to 100 μg / mL, preferably 90 μg / mL to 100 μg / mL, and more preferably 98 μg / mL to 100 μg / mL; The incubation time is 2.5h to 3.5h, preferably 3.3h to 3.5h.

3. The somatic cell chromosome dispersion method according to claim 1, wherein In step S3, at least one of the following methods is selected: The final concentration of colchicine in the cell culture medium system is 7 μg / mL to 13 μg / mL, preferably 9 μg / mL to 11 μg / mL, and more preferably 9.5 μg / mL to 10.5 μg / mL; The processing time is 0.8h to 1.2h, preferably 0.9h to 1.1h.

4. The somatic cell chromosome dispersion method according to any one of claims 1 to 3, wherein In step S1, the step of culturing the body fluid containing somatic cells in a cell culture medium specifically includes the following steps: Somatic fluid containing somatic cells is inoculated into cell culture medium, and phytohemagglutinin is added to a final concentration of 15 μg / mL to 25 μg / mL. The mixture is then incubated at 36℃ to 38℃ in a 4% to 8% CO2 incubator for 65 to 80 hours, preferably 65 to 75 hours. And / or, the body fluid containing somatic cells also contains an anticoagulant; And / or, the body fluid is one of peripheral blood, amniotic fluid, bone marrow, and umbilical cord blood; And / or, the volume ratio of the body fluid to the cell culture medium is 1:3~6, preferably 1:4.5~5.

5.

5. The somatic cell chromosome fragmentation method according to any one of claims 1 to 3, wherein Following step S3, the following steps are also included: Somatic cells treated with colchicine were collected and subjected to hypotonic treatment and fixation treatment in sequence.

6. The somatic cell chromosome dispersion method according to claim 5, wherein In the hypotonic treatment, the patient is treated with a potassium chloride solution with a concentration of 0.01 mol / L to 0.1 mol / L for 25 min to 35 min. And / or, the fixation treatment is performed using a fixative solution, wherein the fixative solution is a mixture of methanol and glacial acetic acid, and the volume ratio of methanol to glacial acetic acid is 2~4:

1.

7. A somatic cell chromosome dispersion kit for chromosome karyotyping, characterized by, The formulation includes cell culture medium, acetaminophen, and colchicine; the final concentration of acetaminophen in the cell culture medium is 60 μg / mL to 110 μg / mL, and the final concentration of colchicine in the cell culture medium is 5 μg / mL to 15 μg / mL.

8. The somatic cell chromosome dispersion kit of claim 7, wherein, The final concentration of the acetaminophen used in the cell culture medium is 60 μg / mL to 100 μg / mL, preferably 90 μg / mL to 100 μg / mL, and more preferably 98 μg / mL to 100 μg / mL. And / or, the final concentration of the colchicine in the cell culture medium is 7 μg / mL to 13 μg / mL, preferably 9 μg / mL to 11 μg / mL, and more preferably 9.5 μg / mL to 10.5 μg / mL.

9. The somatic cell chromosome dispersion kit as described in claim 7, characterized in that, The cell culture medium consists of a basal culture medium supplemented with fetal bovine serum, L-glutamine, penicillin, and streptomycin; The final concentration of the fetal bovine serum is 5%~15% v / v, the final concentration of L-glutamine is 1 mmol / L~10 mmol / L, the final concentration of penicillin is 80 U / mL~120 U / mL, and the final concentration of streptomycin is 80 μg / mL~120 μg / mL.

10. The somatic cell chromosome dispersion kit as described in claim 9, characterized in that, The basal culture medium is RPMI 1640 medium; And / or, the final concentration of the fetal bovine serum is 9%~11% v / v, the final concentration of the L-glutamine is 1 mmol / L~3 mmol / L, the final concentration of the penicillin is 95 U / mL~105 U / mL, and the final concentration of the streptomycin is 95 μg / mL~105 μg / mL.

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