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Composition and kit for rapidly extracting circulating non-consanguineous nucleated cells from peripheral blood, and application thereof

A technology of nucleated cells and compositions, applied in the field of medicine and biology, which can solve the problems of inconvenient application, long time-consuming, low white blood cell removal rate, etc., and achieve the effects of improving efficiency, simplifying operation, and shortening centrifugation time

Pending Publication Date: 2020-07-28
王琦 +1
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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 the immunomagnetic particle method to remove white blood cells, the blood glycophorin-A (glycophorinA) cross-linking method to remove red blood cells, and the immunomagnetic particles and Preliminary attempts to combine density centrifugation, etc., but these methods are time-consuming, have low leukocyte removal rate, poor target cell recovery rate, and require the use of special equipment, which brings inconvenience to application

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Use the composition of the present invention to quickly obtain non-blood circulation nucleated cells from people's blood samples and collect 6ml of people's peripheral blood in any blood collection tube containing anticoagulants (such as EDTA, heparin, or ACD, etc.) (BD , New Jersey, USA). After centrifugation (1000 xg, 15 minutes), plasma proteins were removed.

[0059] After adding 50 microliters of magnetic beads coated with anti-CD14, CD45RO / CD45RA monoclonal antibodies (Invitrogen, California, USA), invert and mix 10 times, and incubate at room temperature for 20 minutes.

[0060] Add 6 ml of cell buffer, and gently transfer the specimen to the top of 3 ml of cell separation medium (the specific gravity of the cell separation medium at 20°C is 1.07260-1.07650 g / ml, and the cell separation medium contains the following components: poly colloidal silica coated with vinylpyrrolidone; polysaccharides and sodium diatrizoate; sugar solution containing dextran; non-ionic...

Embodiment 2

[0084] Utilize the test kit of the present invention to rapidly obtain non-blood circulating nucleated cells from people's blood samples and collect 6ml of people's peripheral blood in any blood collection tube containing anticoagulants (such as EDTA, heparin, or ACD, etc.) ( BD, New Jersey, USA). After centrifugation (1000 xg, 15 minutes), plasma proteins were removed.

[0085] After adding 50 microliters of magnetic beads coated with anti-CD14, CD45RO / CD45RA monoclonal antibodies (Invitrogen, California, USA), invert and mix 10 times, and incubate at room temperature for 20 minutes.

[0086] Add 6 ml of cell buffer, and gently transfer the specimen to the top of 3 ml of cell separation medium (the specific gravity of the cell separation medium at 20°C is 1.07260-1.07650 g / ml, and the cell separation medium contains the following components: poly colloidal silica coated with vinylpyrrolidone; polysaccharides and sodium diatrizoate; sugar solution containing dextran; non-ioni...

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Abstract

The invention provides a composition for uniquely and efficiently separating circulating non-consanguineous nucleated cells from peripheral blood, such as circulating tumour epithelial cells and circulating vascular endothelial cells based on the special density separator centrifugal method combined with a means of removing a consanguineous nucleated cell reagent. The invention relates to a composition for rapidly obtaining circulating non-consanguineous nucleated rare cells from blood. The composition comprises the following components: buffer solution covered with magnetic or non-magnetic immune microspheres or immune particles of an anti-consanguineous nucleated cell surface market antibody; and a cell separation medium.

Description

technical field [0001] The invention belongs to the field of medicine and biology, and relates to a composition and a kit containing nucleated cells. 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 among different types of cells, it cannot be used as a general method for extracting non-blood-related nucleated cells from blood samples. . [0003] A large number of scientific research and clinical experiments recentl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09C12N5/095C12N5/078C12N5/073C12N5/071
CPCC12N5/0693C12N5/0695C12N5/0634C12N5/0603C12N5/069C12N2509/00
Inventor 王琦曾勇
Owner 王琦