Composition and kit for rapid extraction of circulating unrelated nucleated cell from peripheral blood and application thereof

A composition and kit technology, applied in the fields of biochemical equipment and methods, microorganisms, tissue culture, etc., can solve the problems of inconvenient application, low leukocyte removal rate, long time consumption, etc., to simplify operation, shorten centrifugation time, and improve efficiency. Effect

Active Publication Date: 2010-11-10
有限会社林平
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some single methods involved in this method have been reported, such as immunomagnetic particle method to remove white blood cells, blood glycophorin-A (glycophorin A) cross-linking method to remove red blood cells, and immunomagnetic particles that must use special cell separation equipment Preliminary attempts combined with density centrifugation, etc., but these methods take a long time, have low leukocyte removal rate, poor target cell recovery rate, and require special equipment, which brings inconvenience to application

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Utilizing the composition of the present invention to rapidly obtain non-blood circulating nucleated cells from human blood samples

[0056] Collect 10ml of human peripheral blood into any blood collection tube (BD, New Jersey, USA) containing anticoagulant (such as EDTA, heparin, or ACD, etc.). After centrifugation (1000 xg, 15 minutes), plasma proteins were removed.

[0057] After adding 50 ml of hypotonic erythrocyte thawing solution (BD Pharmingen, California, USA), incubate at 4° C. for 30 minutes. The sample specimen was centrifuged again (1000 xg, 15 minutes).

[0058] Resuspend the pelleted cells in 30 mL of buffer. 2 ml of magnetic beads coated with anti-CD45 and / or CD50 monoclonal antibodies (Invitrogen, California, USA) were added thereto, and incubated at room temperature for 30 minutes. The reaction solution is added above the cell separation medium of 20 ml (the specific gravity of the cell separation medium at 20° C. is 1.07260 to 1.07650 g / ml, and the...

Embodiment 2

[0082] Using the kit of the present invention to rapidly obtain non-blood circulating nucleated cells from human blood samples

[0083] Collect 10ml of human peripheral blood into the blood collection tubes carried in the kit containing any of the following anticoagulants, such as EDTA, heparin, or ACD. After centrifugation (1000 xg, 15 minutes), plasma proteins were removed.

[0084] After adding 50 ml of hypotonic erythrocyte thawing solution (BDPharmingen, California, USA) carried in the kit, incubate at 4° C. for 30 minutes. The sample specimen was centrifuged again (1000 xg, 15 minutes).

[0085] Resuspend the deposited cells in 30 mL of the buffer provided in the kit. 2 ml of magnetic beads coated with anti-CD45 and / or CD50 monoclonal antibodies (Invitrogen, California, USA) carried in the kit were added thereto, and incubated at room temperature for 30 minutes. Add the reaction solution above the 20 ml of cell separation medium carried in the kit (the specific gravit...

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Abstract

The invention provides a composition and a kit for rapid and efficient extraction of a circulating unrelated nucleated cell from peripheral blood. The composition for rapid extraction of the circulating unrelated nucleated rare cell from the blood comprises buffer solution, hypotonic erythrocyte breaking solution, magnetic or non-magnetic immune micro-spheres coated with an anti-blood nucleated cell surface marker antibody and a cell separation medium with a certain density. Compared with the common commercial centrifugation medium on the market at present, the composition of the invention can greatly reduce the centrifugation time, and stably improve the cell recovery rate from original 60 plus or minus 20 percent to above 95 percent. When combined with the agent to remove leukocytes andhypotonic erythrocyte breaking means, the composition can be used for effectively and rapidly concentrating and extracting circulating unrelated nucleated rare cells in the peripheral blood, such as epithelial tumor cells and vascular endothelial cells. Therefore, the invention has great practical clinical significance for early diagnosis of tumors and heart diseases.

Description

technical field [0001] The present invention mainly relates to a composition and kit for rapidly extracting circulating non-blood nucleated cells from peripheral blood, and the composition or kit is used in biomedical experiments, or manual clinical inspection operations or for Application in clinical testing operations with the help of automated testing equipment. Background technique [0002] The great clinical significance of extracting and detecting circulating non-blood-related nucleated cells (such as vascular endothelial cells, tumor epithelial cells) in peripheral blood has long been confirmed by people. The current commonly used method for extracting circulating non-blood-related nucleated cells is to use antibodies to combine with special markers on the surface of these cells to achieve the purpose of direct capture. However, this direct capture technique has low sensitivity and poor reproducibility, and because the expression of surface markers varies greatly amo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08C12Q1/02
Inventor 李彦萍
Owner 有限会社林平
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