Blood treating fluid and method for analyzing chromosome karyotype by using same

Abstract

The invention belongs to the field of biological detection, and particularly relates to a blood treating fluid and a method for performing karyotype analysis by using the treating fluid. The treating fluid prepared by the invention comprises a treating fluid A and a treating fluid B. The treating fluid can be used for treating umbilical cord blood of a fetus at the early pregnancy stage, so that the interference of red blood cells can be reduced, and rapid proliferation of lymphocytes can be induced. The umbilical cord blood treated by the treatment liquid is subjected to chromosome karyotype analysis, can be refined into number abnormality or structure abnormality typing, and has important significance for clinical prenatal diagnosis.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and in particular relates to a blood processing liquid and a method for performing chromosome karyotype analysis using the processing liquid. Background Art

[0002] Chromosomal diseases are an important factor causing human birth defects. Therefore, prenatal diagnosis of chromosomal diseases is particularly important. Chromosome testing is an important means of diagnosing chromosomal diseases and conducting genetic research and clinical cytogenetic diagnosis in humans. Although ultrasound diagnostic technology has developed rapidly, many fetal anatomical abnormalities are not discovered until the middle and late stages of pregnancy, resulting in the loss of the best time for diagnosis and treatment. Interventional prenatal diagnosis can diagnose fetal genetic diseases and evaluate intrauterine status in the early stages of pregnancy. Compared with amniotic fluid specimens, umbilical cord blood specimens have the advantages of short culture time, simple methods, easy operation, and fewer pregnancy week restrictions. The use of umbilical cord blood chromosome karyotype analysis can detect patient karyotype abnormalities earlier, which can be refined to numerical abnormalities or structural abnormalities. It is an important basis for the clinical diagnosis of chromosomal diseases and is of great significance for clinical prenatal diagnosis.

[0003] Although cord blood chromosome testing has many advantages, there are a large number of red blood cells in cord blood. Mature red blood cells have no nucleus or organelles, and the cytoplasm is filled with hemoglobin, which hinders chromosome observation after cell culture and affects clinical diagnosis. At the same time, processed cord blood often shows slow cell proliferation and abnormal development during the later lymphocyte culture process. Lymphocyte culture, cell harvesting, drop film and chromosome banding are important links in the preparation of cord blood lymphocyte chromosomes. Any operational error in any link will lead to the failure of chromosome preparation.

[0004] Therefore, a blood treatment fluid capable of reducing red blood cell interference and inducing rapid proliferation of lymphocytes is currently being sought. Summary of the invention

[0005] The object of the present invention is to provide a blood treatment solution, which can not only reduce red blood cell interference but also induce rapid proliferation of lymphocytes.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a blood treatment solution, including a treatment solution A and a treatment solution B;

[0008] The treatment solution A comprises: 0.1-0.2M ammonium chloride, 0.01-0.03M potassium bicarbonate, 0.1-0.2mM EDTA-2Na, 0.8-1.2mM dextran sulfate, 0.05-0.1mM amifostine, 0.1-0.15mM β-carotene, 0.08-0.12mM ectoine, and 0.12-0.18mM sulfaphenazole;

[0009] The treatment solution B comprises: 0.5-0.8 μg / mL OKT3, 80-120 ng / mL IL-2, 0.03-0.06 M glucose, 0.02-0.04 M human transferrin, 15-30 μg / mL vitamin C, and 1.5-2.5 mM glutamine.

[0010] The present invention also provides a method for performing chromosome karyotype analysis using the above-mentioned treatment solution, which specifically comprises the following steps:

[0011] S1, mixing treatment solution A with blood, removing the supernatant and retaining the cell pellet;

[0012] S2, resuspend the cell pellet with treatment solution B, incubate and add culture medium for cultivation;

[0013] S3. 2 h before terminating the culture, add 20 μg / ml colchicine, shake well, and place in a constant temperature box for culture;

[0014] S4, slide preparation: harvest the cultured cells and prepare chromosome slides;

[0015] S5. Microscopic examination: Perform chromosome karyotype analysis on the prepared chromosome specimens.

[0016] Preferably, the volume ratio of the treatment liquid A:blood:treatment liquid B is 10:10:1.

[0017] Preferably, the blood is early pregnancy fetal umbilical cord blood.

[0018] Preferably, the culture medium in step S2 is BC-705 culture medium, and the cell concentration after adding the culture medium is in the range of 1.5×10 6 ~2.0×10 6 cells / mL.

[0019] Preferably, the amount of colchicine added in step S3 is such that the final concentration of colchicine in the solution is 0.1 μg / ml.

[0020] Preferably, the chromosome preparation process in step S4 includes subjecting the harvested cells to hypotonic treatment, pre-fixation, centrifugation, fixation, centrifugation, re-fixation, centrifugation, preparation of cell suspension, drop-slicing and staining.

[0021] More preferably, the fixing solution used in the pre-fixation process includes methanol and glacial acetic acid, and the volume ratio of methanol to glacial acetic acid is 3:1.

[0022] More preferably, the dye solution used in the dyeing process includes Giemsa solution stock solution and phosphate buffer with a pH of 6.8, and the mass ratio of the Giemsa solution stock solution to the phosphate buffer with a pH of 6.8 is 1:9.

[0023] The present invention also provides the use of the above-mentioned treatment solution or the above-mentioned method for performing chromosome karyotype analysis in chromosome karyotype analysis of umbilical cord blood of a fetus in early pregnancy.

[0024] Beneficial effects of the present invention:

[0025] The blood treatment fluid of the present invention and the method for chromosome karyotype analysis using the treatment fluid can destroy mature red blood cells, enrich white blood cells in the blood and efficiently proliferate lymphocytes without causing damage to lymphocytes in umbilical cord blood, and can be used for subsequent chromosome preparation to ensure normal karyotype, which is of great significance to clinical medical examinations. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0027] Figure 1 The flow cytometry results of blood samples before and after treatment with treatment solution A;

[0028] Figure 2 This is the phase diagram of chromosome division;

[0029] Figure 3 This is a chromosome karyotype diagram. DETAILED DESCRIPTION

[0030] The present invention provides a blood treatment solution, which comprises a treatment solution A and a treatment solution B. The treatment solution A comprises: 0.1-0.2M ammonium chloride, 0.01-0.03M potassium bicarbonate, 0.1-0.2mM EDTA-2Na, 0.8-1.2mM dextran sulfate, 0.05-0.1mM amifostine, 0.1-0.15mM β-carotene, 0.08-0.12mM ectoine, and 0.12-0.18mM sulfaphenazole; preferably 0.15M ammonium chloride, 0.01M potassium bicarbonate, 0.1mM EDTA-2Na, 1mM dextran sulfate, 0.05mM amifostine, 0.1mM β-carotene, 0.1mM ectoine, and 0.15mM sulfaphenazole.

[0031] Treatment solution B includes: 0.5-0.8 μg / mL OKT3, 80-120 ng / mL IL-2, 0.03-0.06 M glucose, 0.02-0.04 M human transferrin, 15-30 μg / mL vitamin C, and 1.5-2.5 mM glutamine; preferably 0.5 μg / mL OKT3, 100 ng / mL IL-2, 0.05 M glucose, 0.02 M human transferrin, 20 μg / mL vitamin C, and 2 mM glutamine.

[0032] The present invention uses the blood processing fluid to prepare a method for chromosome karyotype analysis, which specifically includes three steps: umbilical cord blood processing and cell culture, slide preparation and microscopic examination.

[0033] The volume ratio of treatment solution A, cord blood and treatment solution B during cord blood processing and cell culture is 10:10:1. Treatment solution A is mixed with the sample and then centrifuged, and the supernatant is discarded. The precipitated material is resuspended in the liquid using DPBS, which can maintain a stable pH value and does not affect the physiological functions of cells and molecules.

[0034] Repeat the centrifugation and supernatant removal process three times and keep the cell pellet. Resuspend the cell pellet in solution B, take samples and count them. After incubation for 20 minutes, add a certain amount of BC-705 medium to make the cell density 1.5×10 6 ~2.0×10 6 cells / mL, and transfer to a cell culture dish or cell culture flask for static culture.

[0035] Colchicine was added 2 hours before terminating the culture. Colchicine can inhibit mitosis, destroy the spindle, and cause chromosomes to stop in metaphase. This abnormal division caused by colchicine is called colchicine mitosis. In such mitosis, although the chromosomes split longitudinally, the cells do not divide and cannot form two daughter cells, thus causing the chromosomes to double, thereby accumulating more dividing cells that stop in metaphase.

[0036] The process of slide preparation includes harvesting cells, hypotonic treatment, pre-fixation, centrifugation, fixation, centrifugation, re-fixation, centrifugation, preparation of cell suspension, drop-slide and staining.

[0037] During the pre-fixation process, methanol and glacial acetic acid are added as fixatives to fix and preserve cell and tissue samples. The methanol in the methanol and glacial acetic acid fixative can dissolve lipids in the cell membrane and fix the cell membrane. At the same time, the acetic acid in the fixative can react with some amino acid residues formed by proteins to produce acetate compounds, thereby fixing the nuclear proteins; it can also better show the location and number of chromosomes during the staining process.

[0038] The staining process utilizes the Giemsa staining method, whose basic components are eosin and methylene blue, to improve the quality of the dye, so that the cell nucleus is better colored and the structure is displayed more clearly.

[0039] Microscopic examination, using an optical microscope to analyze the chromosome karyotype, can be used to determine the risk of certain diseases. Each human somatic cell has 46 chromosomes, which can be divided into 22 pairs of autosomes and one pair of sex chromosomes based on the length of the chromosomes and the position of the centromere. Males have 46, XY; females have 46, XX.

[0040] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0041] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

[0042] Example 1 Preparation of treatment solution

[0043] A processing solution for preparing umbilical cord blood chromosomes, comprising 5 mL of processing solution A and 0.5 mL of processing solution B.

[0044] S1: Treatment solution A includes: 0.2 M ammonium chloride, 0.15 M potassium bicarbonate, 0.2 mM EDTA-2Na, 0.8 mM dextran sulfate, 0.1 mM amifostine, 0.15 mM β-carotene, 0.08 mM ectoine, 0.18 mM sulfaphenazole, and double distilled water is added to make up to 5 mL;

[0045] S2: Treatment solution B includes: 0.8 μg / mL OKT3, 80 ng / mL IL-2, 0.06 M glucose, 0.04 M human transferrin, 15 μg / mL vitamin C, 1.5 mM glutamine, and double distilled water is added to make up to 0.5 mL.

[0046] Example 2 Preparation of treatment solution

[0047] A processing solution for preparing umbilical cord blood chromosomes, comprising 5 mL of processing solution A and 0.5 mL of processing solution B.

[0048] S1: Treatment solution A: 0.15 M ammonium chloride, 0.01 M potassium bicarbonate, 0.1 mM EDTA-2Na, 1 mM dextran sulfate, 0.05 mM amifostine, 0.1 mM β-carotene, 0.1 mM ectoine, 0.15 mM sulfaphenazole, add double distilled water to make up to 5 mL;

[0049] S2: Treatment solution B: 0.5 μg / mL OKT3, 100 ng / mL IL-2, 0.05 M glucose, 0.02 M human transferrin, 20 μg / mL vitamin C, 2 mM glutamine, add double distilled water to make up to 0.5 mL.

[0050] Example 3 Chromosome Karyotype Analysis

[0051] (1) Umbilical cord blood processing and cell culture:

[0052] The treatment solution was sterilized by filtration with a 0.22 μm filter membrane before use. Treatment solutions A and B were prepared according to the preparation method of the treatment solution in Example 2. Treatment solution A was mixed with umbilical cord blood in a volume ratio of 1:1 and then centrifuged at 500×g for 10 minutes, the supernatant was discarded, and the cell pellet was retained after resuspending with 1 mL of DPBS and centrifuged at 500×g for 10 minutes. The operation was repeated three times.

[0053] The results of treatment with treatment solution A showed that the number of red blood cells decreased significantly after treatment with solution A, and the effect was obvious. At the same time, the number of white blood cells was maintained at a certain level, which was sufficient for subsequent culture. Figure 1 As shown, treatment solution A is effective and can remove most red blood cells and cell debris.

[0054] Table 1 Comparison of cells before and after treatment with treatment solution A

[0055]

[0056]

[0057] The volume ratio of the cell pellet to the treatment solution B was 1:10 (cord blood volume), and samples were taken for counting. After incubation for 20 min, a certain amount of BC-705 medium (cell density was 1.5×106 ~2.0×10 6 cells / mL), transferred to a cell culture dish or cell culture flask, and cultured at 37°C for 72 h.

[0058] 2 hours before terminating the culture, add 20 μg / ml colchicine to a final concentration of 0.1 μg / ml. After gently shaking, place the cells in a constant temperature incubator and continue culturing for 2 to 3 hours to accumulate more cells that have stopped dividing in the metaphase.

[0059] (2) Production:

[0060] ① Harvesting cells: transfer the cultured cells to an appropriate centrifuge tube, centrifuge at 500×g for 10 min, and discard the supernatant; resuspend in 1 mL DPBS and centrifuge at 500×g for 10 min, and discard the supernatant. Repeat this operation three times and retain the cell pellet.

[0061] ②Hypotonic treatment: Add 8 ml of 0.075 mol / L KCl pre-warmed at 37°C, pipette and vortex repeatedly, and incubate in a 37°C constant temperature heating apparatus for 25 min.

[0062] ③ Pre-fixation: Add 1 ml of freshly prepared fixative (methanol: glacial acetic acid = 3:1) and mix gently.

[0063] ④ Centrifugation: 500×g, centrifuge for 10 min, discard the supernatant.

[0064] ⑤ Fixation: Slowly add 8 ml of fixative along the wall of the centrifuge tube, mix gently, and fix for 10 minutes.

[0065] ⑥ Centrifugation: 500×g, centrifuge for 10 minutes, and aspirate the supernatant.

[0066] ⑦ Re-fix: add 8 ml of fixative, mix well, and fix for 10 minutes.

[0067] ⑧Centrifugation: 500×g, centrifuge for 10 min, and aspirate the supernatant.

[0068] ⑨ Prepare cell suspension: Add appropriate amount of fixative according to the number of cells and mix thoroughly to make cell suspension.

[0069] ⑩ Dropping: Take out the slide that has been soaked in ice water in advance, use a pipette to absorb the mixed cell suspension and drop it on the slide at a distance of 20 cm from the ice. Drop about 2 to 3 drops of cell suspension on each slide to make the cells better dispersed.

[0070] Staining: After the specimen is dried, it can be stained with staining solution (1 part Giemsa solution, 9 parts pH 6.8 phosphate buffer) for 15 minutes, rinse with tap water (rinse from the back) and dry.

[0071] (3) Microscopic examination:

[0072] Each human somatic cell has 46 chromosomes, which can be divided into 22 pairs of autosomes and one pair of sex chromosomes according to the length of the chromosomes and the position of the centromere. Males have 46, XY; females have 46, XX.

[0073] The results of karyotype analysis were as follows Figure 2-3 As shown, the chromosomes treated by this method have obvious morphological and structural characteristics and normal number.

[0074] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A blood treatment fluid, characterized in that: including a treatment liquid A and a treatment liquid B; The treatment solution A comprises: 0.1-0.2M ammonium chloride, 0.01-0.03M potassium bicarbonate, 0.1-0.2mM EDTA-2Na, 0.8-1.2mM dextran sulfate, 0.05-0.1mM amifostine, 0.1-0.15mM β-carotene, 0.08-0.12mM ectoine, and 0.12-0.18mM sulfaphenazole; The treatment solution B comprises: 0.5-0.8 μg / mL OKT3, 80-120 ng / mL IL-2, 0.03-0.06 M glucose, 0.02-0.04 M human transferrin, 15-30 μg / mL vitamin C, and 1.5-2.5 mM glutamine.

2. A method for chromosome karyotype analysis using the treatment solution of claim 1, characterized in that: The following steps are involved: S1, mixing treatment solution A with blood, removing the supernatant and retaining the cell pellet; S2, resuspend the cell pellet with treatment solution B, incubate and add culture medium for cultivation; S3. 2 h before terminating the culture, add 20 μg / ml colchicine, shake well, and place in a constant temperature box for culture; S4, slide preparation: harvest the cultured cells and prepare chromosome slides; S5. Microscopic examination: Perform chromosome karyotype analysis on the prepared chromosome specimens.

3. The method according to claim 2, characterized in that: The volume ratio of the treatment liquid A:blood:treatment liquid B is 10:10:

1.

4. The method according to claim 2, characterized in that: The blood is umbilical cord blood of a fetus in early pregnancy.

5. The method according to claim 2, characterized in that: The culture medium in step S2 is BC-705 culture medium, and the cell concentration range after adding the culture medium is 1.5×10 6 ~2.0×10 6 cells / mL.

6. The method according to claim 2, characterized in that: The amount of colchicine added in step S3 is such that the final concentration of colchicine in the solution is 0.1 μg / ml.

7. The method according to claim 2, characterized in that: The chromosome preparation process in step S4 includes hypotonic treatment of the harvested cells, pre-fixation, centrifugation, fixation, centrifugation, re-fixation, centrifugation, preparation of cell suspension, drop-slicing and staining.

8. The chromosome preparation process according to claim 7, characterized in that: The fixing solution used in the pre-fixation process includes methanol and glacial acetic acid, and the volume ratio of the methanol to the glacial acetic acid is 3:

1.

9. The chromosome preparation process according to claim 7, characterized in that: The dye solution used in the dyeing process includes Giemsa solution stock solution and phosphate buffer solution with a pH value of 6.8, and the mass ratio of the Giemsa solution stock solution to the phosphate buffer solution with a pH value of 6.8 is 1:

9.

10. Use of the treatment solution of claim 1 or the method of any one of claims 2 to 9 in chromosome karyotype analysis of umbilical cord blood of a fetus in early pregnancy.

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