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Method for detecting non-humoral rare karyotes, and kit thereof

A technology of humoral cells and nucleated cells, which is applied in the field of detection of non-humoral rare nucleated cells, can solve the problems of repeated positioning operation difficulties, easy loss, damaged cells, etc., and achieve the effect of high detection sensitivity

Active Publication Date: 2014-08-27
CYTTEL BIOSCI BEIJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0013] In view of the above-mentioned use of epithelial cell markers alone to detect non-humoral rare nucleated cells is likely to produce false positive, weak positive or false negative results, and FISH alone cannot determine whether it is lymphocyte mutation or impurity interference, and separate use of FISH and Immunofluorescence treatment is easy to lose and damage cells, and repeated positioning operations are difficult, and FISH treatment and observation of FISH results are performed first, which may easily cause technical problems such as degradation of immune markers. The present invention proposes a detection method for rare cells, which is used in combination with Leukocyte markers are used as humoral cell surface markers, and tumor-specific nucleic acid mutations are used as FISH detection probes, which can not only observe two signals at the same time, but also avoid cell loss and damage, and because the observation of one result is omitted, It can avoid the degradation of cell surface antigen markers to the greatest extent, improve the sensitivity and specificity of non-humoral rare nucleated cells detection, on the other hand, it can improve the reading speed and avoid repeated positioning

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  • Method for detecting non-humoral rare karyotes, and kit thereof
  • Method for detecting non-humoral rare karyotes, and kit thereof
  • Method for detecting non-humoral rare karyotes, and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0149] The results of different processing sequences of FISH and CD45

[0150] Methods: Four tubes of peripheral blood collected from the same person were treated with FISH and CD45 in different order.

[0151] Experimental group 1: CEP8 was used as a FISH probe, and CD45 was used as a leukocyte surface marker. On the day of blood collection, randomly select a tube of blood for negative enrichment (the enrichment product is the leukocyte depletion kit of our company), smear the specimen on a glass slide, and place it in an oven at 37°C until it is completely dry. Afterwards, 1% paraformaldehyde was fixed for 8 minutes, and the cells were processed by FISH first and then CD45. The FISH processing steps are as follows: soak in 2×SSC at 37°C for 15 minutes, dehydrate in 75%, 85%, and absolute ethanol for 5 minutes each, add 10ul probes, cover with a cover slip, and hybridize in a DAKO hybridization instrument for 20 minutes. Afterwards, wash with 50% formamide for 15 min, and 2...

Embodiment 2

[0160] Sensitivity test of the method of the present invention in a cell line model

[0161] Peripheral blood was used as the body fluid of this test, and the leukocytes were humoral nucleated cells, and CD45 was selected as the humoral cell surface marker. The A549 cell line with genetic characteristics of trisomy 8 was selected as the non-humoral rare nucleated cells, and some cells of this cell line had three homologous chromosomes 8. By labeling it with a fluorescent marker, the recovery rate of this method is tested to identify its sensitivity. The centromere probe of Vysis Company was selected as the probe for chromosome 8: CEP8 (orange).

[0162] This test was processed first with FISH and then with CD45.

[0163] Take 1ml of normal human peripheral blood, remove the plasma and lyse the red blood cells, count the white blood cells with a hemocytometer under an optical microscope, dilute with PBS and resuspend to a concentration of 142 cells / ul. Take 8 parts of the di...

Embodiment 3

[0174] False positive test of the present invention in multiple normal persons

[0175] 22 cases of normal people were included in the false positive test, and the leukocyte depletion kit of our company was used for negative enrichment, with 2 smears for each. One of them is used for the test of the method of the present invention, and the other is used for the test of FISH.

[0176] One of them was fixed with 1% paraformaldehyde for 8 minutes by this method, and then processed in the order of FISH and then CD45, and the other was treated only with FISH as a comparison. No. 8 centromere probe (Vysis, orange) was used for screening. The processing steps of this method are as follows: soak the sample in 2×SSC at 37°C for 15 minutes, and dehydrate it step by step with 75%, 85%, absolute ethanol, each step is 4 minutes. Add 10ul probes, cover with a cover slip, put in a hybridization instrument (DAKO) for denaturation for 5 minutes, and hybridize for 20 minutes. Then peel off t...

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Abstract

The invention discloses a new method for detecting non-humoral rare karyotes. Non-humoral rare karyotes in a biological body fluid are obtained through many means, and the combined use of the antibody of a humoral cell surface marker and the specific probe of the non-humoral rare karyotes is carried out to discriminate the enriched rare karyotes. Compared with commercial discriminating methods existing in the present market, the method disclosed in the invention well combines a fluorescent in situ hybridization (FISH) method for processing the non-humoral rare karyotes with the immunofluorescence dyeing for processing humoral rare karyotes, realizes the systemic associative processing of a same specimen sheet, and can realize the intelligible observation of two processing results.

Description

technical field [0001] The present invention relates to a detection method of non-humoral rare nucleated cells, which comprises the combined use of antibody of humoral cell surface markers and specific probe treatment of non-humoral rare nucleated cells after re-fixing the sample samples to be tested. The present invention also relates to kits comprising i.) antibodies to humoral cell surface markers; ii.) specific probes for non-humoral rare nucleated cells; and iii.) refixatives for use in the present invention Uses of the method of the invention, and detection systems for implementing the method of the invention. Background technique [0002] In recent years, more and more reports are keen on the research of circulating tumor cells (CTC), circulating endothelial cells (CEC), tumor stem cells, etc., and the results mostly cover solid tumors such as breast cancer, colorectal cancer, and prostate cancer (Joost, Cytometry Part A, 75A, 2009; Shaffer DR, Clin Cancer Research,...

Claims

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Application Information

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IPC IPC(8): G01N33/53G01N21/64
CPCG01N33/57407G01N33/57484G01N2333/70525G01N2333/70589
Inventor 詹厅
Owner CYTTEL BIOSCI BEIJING
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