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Reagents and methods for enriching circulating rare cells

A rare cell and nucleated cell technology, applied in the field of oncology, can solve problems such as high detection cost, complicated device operation, and inability to analyze samples

Active Publication Date: 2022-05-06
GENOSABER BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, none of the current single leukocyte markers can label and remove all leukocytes. Therefore, although the extraction rate of the remaining CRC is high, the purity is not ideal, usually below 0.1%, and the genotype analysis of the sample cannot be performed directly.
[0008] Therefore, the current CRC detection technology still has the defects of low detection sensitivity, few tumor types, complicated device operation and high detection cost.

Method used

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  • Reagents and methods for enriching circulating rare cells
  • Reagents and methods for enriching circulating rare cells
  • Reagents and methods for enriching circulating rare cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Embodiment 1, the preparation of CRC microfluidic detection system

[0102] The core of the hardware part of the CRC microfluidic detection system of the present invention is a miniature metal conductor. By receiving the sorting signal of the CRC fluorescence detection component 5, the microthermal bubbles emitted from the microfluidic generator 6 are triggered to drive the microthermal bubbles in the cavity of the jet unit. The fluid ejected out of the cavity pushes the CRC just past the sorting node into the positive channel.

[0103] Its preparation process is as figure 2 As shown, at first the substrate 8 is provided, and the metal conductor (such as aluminum or copper) that the micro heat bubble occurs is processed on a silicon or glass substrate by photolithography technology, and a protective layer (such as silicon dioxide) is deposited thereon ( figure 2 Steps a), b)). At the same time, process the microfluidic structure on another glass cover slip 9, and bo...

Embodiment 2

[0105] Example 2, Screening of white blood cell fluorescently labeled antibodies

[0106] For the mixed sample of CRC and white blood cells after pretreatment, the key is how to effectively use multiple markers to remove white blood cells to the greatest extent and improve the recognition rate and specificity of CRC. The present inventors investigated the labeling efficiency of leukocytes by multiple CD fluorescent antibodies using flow cytometry.

[0107] Take two 3mL healthy human blood samples, the experimental steps are as follows:

[0108] (1) Removal of red blood cells: Dilute the whole blood with an equal amount of PBS and slowly add it to a centrifuge tube with an equal volume of histopaque, and centrifuge at 2000rpm for 30 minutes; after the blood is centrifuged, obtain the cell layer between the plasma and the red blood cell layer;

[0109] (2) Washing: Gently remove the cells from the cell layer of (1), add an equal amount of PBS to wash, centrifuge at 600g for 15m...

Embodiment 3

[0119] Embodiment 3, the establishment of the CRC microfluidic detection system of the present invention

[0120] like figure 1 , Establish the microfluidic detection device of the present invention. The microfluidic detection device includes a microfluidic structure 7, which includes a single cell channel that allows single cells to pass through at a stable flow rate. The single cell channel starts from the sample inlet 1, and there are multiple single cell channels downstream of it to allow single cells to pass through. For the shunting of cells, one channel leads to the CRC collection tube 3, and the other or more channels lead to the remaining cell collection tube 2; the fluorescence detection component 5 can identify each cell according to different fluorescent signals, and quickly confirm its identity. Determine whether to sort them in the next step; the micro-fluidic generator 6, which includes a miniature metal conductor, receives the sorting signal from the fluoresce...

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Abstract

The present invention relates to reagents and methods for enriching circulating rare cells (CRC). The present invention discloses a method for separating leukocytes in blood by using a preferred combination of CD markers to enrich CRC. The method is a negative sorting method. The method of the present invention can greatly improve the accuracy of cell identification, realize a breakthrough in the sorting of high-purity CRC, and the obtained samples are more conducive to being directly applied to subsequent genome analysis. At the same time, the invention also provides a microfluid detection device applied to the method.

Description

technical field [0001] The invention belongs to the field of oncology, and more specifically, the invention relates to a reagent for enriching circulating rare cells, its use, and a method for enriching circulating rare cells. Background technique [0002] At present, the formulation of individualized cancer treatment plans relies on tumor information in tissue samples, but it is difficult to obtain tissue samples in clinical practice, especially during treatment and in the early stage of cancer metastasis. Therefore, finding substitutes for tissue samples is the most urgent requirement for cancer treatment monitoring and drug resistance mechanism research. [0003] Circulating tumor cells (CTCs) are cells released from primary tumors that carry tumor-specific biological information. Due to the convenience and timeliness of peripheral blood detection, CTC, as an easy-to-obtain tumor information carrier, can provide a sample for dynamic monitoring of changes in tumor biologi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/533
Inventor 杨国华
Owner GENOSABER BIOTECH CO LTD
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