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Method for separating fetal nucleated red blood cells in maternal peripheral blood

A technology for pregnant women's peripheral blood and nuclear red blood cells, which can be applied to embryonic cells, blood/immune system cells, animal cells, etc., and can solve problems such as stability and accuracy that need to be improved and clinical difficulties

Inactive Publication Date: 2014-02-26
邯郸市康业生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, density gradient centrifugation, monoclonal antibody labeling combined with flow cytometry are still in the experimental stage, stability and accuracy need to be improved, and it is difficult to be widely used in clinical practice.

Method used

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  • Method for separating fetal nucleated red blood cells in maternal peripheral blood

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Effect test

Embodiment 1

[0034] A method for isolating fetal nucleated erythrocytes in the peripheral blood of pregnant women, comprising the following steps:

[0035] 1. Enrichment of nucleated cells

[0036] (1) Aseptically collect 10 mL of whole blood in 15 mL vacuum EDTA anticoagulant blood collection tubes to avoid hemolysis.

[0037] (2) Centrifuge at room temperature at 800g for 10 minutes, discard the supernatant (can be used for serum DNA extraction), and collect the cell pellet.

[0038] (3) Add an equal volume of 37°C preheated RPMI1640 cell culture medium to the above cell pellet. The RPMI1640 cell culture medium contains fetal bovine serum with a mass fraction of 10%, and mix gently and thoroughly to obtain a diluted cell suspension .

[0039] (4) In the centrifuge tube pre-prepared with 3 mL of cell separation solution, slowly add 4 mL of diluted cell suspension on the layered liquid surface along the wall of the tube with a dropper;

[0040] The composition of the cell separation liq...

Embodiment 2

[0050] A method for isolating fetal nucleated erythrocytes in the peripheral blood of pregnant women, comprising the following steps:

[0051] 1. Enrichment of nucleated cells

[0052] (1) Aseptically collect 10 mL of whole blood in 15 mL vacuum EDTA anticoagulant blood collection tubes to avoid hemolysis.

[0053] (2) Centrifuge at room temperature at 800g for 10 minutes, discard the supernatant (can be used for serum DNA extraction), and collect the cell pellet.

[0054] (3) Add an equal volume of 37°C preheated RPMI1640 cell culture medium to the above cell pellet. The RPMI1640 cell culture medium contains fetal bovine serum with a mass fraction of 10%, and mix gently and thoroughly to obtain a diluted cell suspension .

[0055] (4) In the centrifuge tube pre-prepared with 3 mL of cell separation solution, slowly add 4 mL of diluted cell suspension on the layered liquid surface along the wall of the tube with a dropper;

[0056] The composition of the cell separation liq...

Embodiment 3

[0066] A method for isolating fetal nucleated erythrocytes in the peripheral blood of pregnant women, comprising the following steps:

[0067] 1. Enrichment of nucleated cells

[0068] (1) Aseptically collect 10 mL of whole blood in 15 mL vacuum EDTA anticoagulant blood collection tubes to avoid hemolysis.

[0069] (2) Centrifuge at room temperature at 800g for 10 minutes, discard the supernatant (can be used for serum DNA extraction), and collect the cell pellet.

[0070] (3) Add an equal volume of 38°C preheated RPMI1640 cell culture medium to the above-mentioned cell sediment. The RPMI1640 cell culture medium contains fetal bovine serum with a mass fraction of 10%, and mix gently and thoroughly to obtain a diluted cell suspension .

[0071] (4) In the centrifuge tube pre-prepared with 3 mL of cell separation solution, slowly add 4 mL of diluted cell suspension on the layered liquid surface along the wall of the tube with a dropper;

[0072] The composition of the cell se...

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Abstract

The invention discloses a method for separating fetal nucleated red blood cells in maternal peripheral blood. The method comprises the following steps: (a) enriching nucleated cells, namely, (1) collecting whole blood, (2) centrifuging, and collecting cell precipitates, (3) preparing cell suspension, namely adding a cell culture medium to the cell precipitates, and uniformly mixing so as to prepare the dilute cell suspension, (4) putting a cell separating medium in a centrifugal tube in advance, and adding the dilute cell suspension to a layering liquid surface, (5) centrifuging so as to obtain a cell layer, and (6) sucking up the cell layer, adding Hank, s liquid, uniformly mixing, centrifuging at a room temperature, and washing cells, and (7) resuspending the cells; and (b) screening the fetal nucleated cells in the maternal blood, to be specific, (9) separating out the fetal nucleated red cells, and (10) carrying out quadruple morphology identification on nucleated red cell pictures obtained by sorting, and collecting the nucleated red cells meeting the standards for extracting the fetal whole genome DNA (deoxyribonucleic acid). According to the method, the purity and the recovery rate of the fetal nucleated red blood cells obtained by the separation and purification of the maternal peripheral blood are high.

Description

technical field [0001] The invention relates to a method for isolating fetal nucleated red blood cells and belongs to the technical field of genetic engineering. Background technique [0002] Birth defects refer to various abnormalities in body structure, intelligence or metabolism that exist at birth. my country is one of the countries with a high incidence of defective newborns in the world. 5% of the 20 million newborns born in the country each year have birth defects, and a defective child is born every 30 seconds. Birth defects can be caused by genetic factors such as chromosomal aberrations and gene mutations or environmental factors, or by the interaction of these two factors or other unknown reasons. Existing scientific research results show that gene mutations play an important role in the occurrence of birth defects and genetic diseases, so prenatal screening of fetal chromosomes and DNA is an important means to reduce birth defects and genetic diseases. [0003]...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073C12N5/078
Inventor 吴超群徐晓丽
Owner 邯郸市康业生物科技有限公司
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