Improved fluorescence in-situ hybridization method and application thereof

A fluorescence in situ hybridization technology, applied in the field of improved fluorescence in situ hybridization, can solve the problems of too much influence of subjective human factors, difficult to distinguish cells, and poor prognosis.

Inactive Publication Date: 2017-03-08
AFFILIATED HOSPITAL OF NANTONG UNIV
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  • Summary
  • Abstract
  • Description
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Problems solved by technology

[0003] In the past, the evaluation indicators of diagnosis, treatment and prognosis mainly relied on the patient's clinical manifestations, peripheral blood, bone marrow cytomorphology, bone marrow biopsy, conventional cytogenetic (CC) studies, immune typing, etc. There are the following problems: First, although The quality and quantity of cell changes are an important basis for morphological diagnosis, but it is difficult to clearly distinguish between atypical prolymphocytes, prolymphocytes, myeloblasts and cells expressing myeloid and lymphoid markers at the same time; second, CC technology can only analyze metaphase cells, which are affected by the cell cycle and the number and quality of chromosomal division, which may lead to f...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: a kind of improved fluorescence in situ hybridization technique, comprises the following steps:

[0023] (1-1) Aseptically extract 2~5ml of the patient's bone marrow, and adjust the number of mononuclear cells in the clean bench to 1~2×10 6 / ml, injected with 1640 culture solution, cultured at 37°C for 24 hours;

[0024] (1-2) Add colcemid application solution before terminating the culture, then prepare slices by hypotonicity, pre-fixation, fixation, and air-drying, and store the rest of the cell suspension at -20°C;

[0025] (1-3) Take out the specimens stored at -20°C, replace them with fresh fixative solution, soak in 2×SSC at 37°C for 30 minutes, and put them in ethanol with volume fractions of 70%, 85%, and 100% at room temperature Gradient dehydration, each gradient dehydration 2min;

[0026] Wherein, the fixative includes methanol and glacial acetic acid, and the weight ratio of methanol and glacial acetic acid is 3:1.

[0027] (1-4) Denature at...

Embodiment 2

[0034] Embodiment 2: a kind of improved fluorescent in situ hybridization technique, comprises the following steps:

[0035] (2-1) Aseptically extract 2~5ml of the patient's bone marrow, and adjust the number of mononuclear cells in the clean bench to 1~2×10 6 / ml, injected with 1640 culture solution, cultured at 37°C for 24 hours;

[0036] (2-2) Before terminating the culture, add colcemid application solution, then make slices by hypotonicity, pre-fixation, fixation, and air-drying, and store the rest of the cell suspension at -20°C;

[0037] (2-3) Take out the specimens stored at -20°C, replace them with fresh fixative solution, soak in 2×SSC at 37°C for 31.5 minutes, and put them in ethanol with volume fractions of 70%, 85%, and 100% at room temperature Lower gradient dehydration, each gradient dehydration 2min;

[0038] Wherein, the fixative includes methanol and glacial acetic acid, and the weight ratio of methanol and glacial acetic acid is 3:1.

[0039] (2-4) Denatu...

Embodiment 3

[0047] Example 3: Application of an improved fluorescence in situ hybridization method in the preparation of a detection kit for lymphoid leukemia gene deletion, such as for detecting the deletion of p53 gene in patients with lymphoid leukemia.

[0048] 19 patients with acute lymphoid leukemia (ALL) and 62 patients with chronic lymphocytic leukemia (CLL) were detected and analyzed using the Fluorescence in Situ hybridization (FISH) technique of the present invention. The former uses specific probes BCR / ABL, MLL, IHG, C-MYC, TEL / AML1, and the latter uses D13S25(13q14.3), RB1(13q14), ATM(11q22.3), p53(17p13), CSP12 Detection analysis.

[0049] Among the 81 cases of lymphoid leukemia: molecular genetic abnormalities were detected in 49 cases, accounting for 58.02% (47 / 81). Of the 19 ALL patients, 7 had one or more molecular genetic abnormalities, accounting for 36.84% (7 / 19); 3 had two or more molecular genetic abnormalities, accounting for 15.79% (3 / 19) ), among which 4 cases ...

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Abstract

The invention provides an improved fluorescence in-situ hybridization method and an application thereof. The improved fluorescence in-situ hybridization method comprises the following steps: processing a specimen, conducting denaturation, processing a probe, conducting hybridization and conducting counter-staining. According to the improved fluorescence in-situ hybridization method provided by the invention, by conducting observation by virtue of a fluorescence microscope and calculating fluorescence hybridization signals of cells, conducting precise chromosome analysis on 81 patients with lymphoproliferative diseases and judging gene deletions and molecular genetic abnormalities of the patients, the following conclusions are obtained: molecular genetic analysis is conducive to diagnosis and differential diagnosis of lymphoid leukemia, and moreover, the molecular genetic analysis also serves as an important index for monitoring disease remission and recurrence and for judging a curative effect in the clinical field. On the basis of chromosomal karyotype analysis, the proper fluorescence in-situ hybridization probe and method are selected to conduct precise chromosome analysis on most lymphoid leukemia patients, and the various molecular genetic abnormalities are related to prognosis of the patients.

Description

technical field [0001] The invention relates to the technical fields of cytogenetics and molecular biology, in particular to an improved fluorescence in situ hybridization method and its application. Background technique [0002] In 2001, WHO carried out a new classification of lymphoid hematopoietic tissue tumors. Currently, clinically, lymphoproliferative diseases mainly refer to lymphocytic leukemia and some malignant lymphomas. Lymphocytic leukemia is a clonal malignant proliferation disease originating from B or T lineage lymphocytes, mainly including acute lymphocytic leukemia (ALL) and chronic lymphocytic leukemia (CLL). The former is characterized by abnormal proliferation and accumulation of immature prolymphocytes in blood, bone marrow, and lymphoid tissues. The latter is characterized by a chronic malignant proliferation of immunocompromised mature small lymphocytes in the blood, bone marrow, and lymphoid tissues, eventually leading to failure of normal hematopoi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6841C12Q1/6886C12Q2600/118C12Q2600/156C12Q2563/107
Inventor 姜胜华曹鑫刘红杨力陆伟林赠华孙峰宋国齐温慧灵
Owner AFFILIATED HOSPITAL OF NANTONG UNIV
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