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Blood cell separation system

A blood cell and system technology, applied in analytical materials, biological material analysis, material inspection products, etc.

Inactive Publication Date: 2005-05-11
NETECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Also, in order to understand the variation of chromosomes, etc. in the separation and purification of nucleated erythroblasts by this method, they must be checked by the FISH (fluorescence in situ hybridization) method, etc., but this test is performed in preparation The process of sampling produces practical problems that need to be overcome

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1: Primary Separation Using Density Centrifugation

[0067] Histopaque (Sigma) was obtained and used as a density centrifugation reagent to which sodium diatrizoate was added and 6 types of density gradient fluids were prepared with specific gravity adjusted to 1.077-1.105. 7 cc of venous blood was obtained from pregnant women at 10-20 weeks of pregnancy, and then centrifuged in each density gradient fluid for 30 minutes (20° C., 1500 rpm). The collected cells surrounding the boundary between the density gradient fluid and the plasma fraction (upper layer) are recovered and centrifuged with a biological buffer to obtain a crude fraction free of most non-nucleated red blood cells and platelets.

[0068] Samples that were initially purified at various densities were subjected to secondary separations using the carbohydrate-lectin method. As a substrate, a plastic slide chamber (2-well, product of Nalgenunc) was used. The complex carbohydrate polymer coated on th...

Embodiment 2

[0071] Example 2: Additional isolation by panning

[0072] Plastic slide chambers (4 wells, product of Nalgenunc) were treated with FCS or 0.01 wt% aqueous solution of complex glycopolymers (PV-glyco) (product of Netech). As complex glycopolymers, those containing glucose, maltose, gluconic acid, N-acetylglucosamine, mannose, lactose or melibiose structures are used.

[0073] The recovered postnatal cord blood was subjected to density gradient centrifugation using Histopaque (d, 1.095) according to standard methods, and cells agglutinated near the boundary between Histopaque and plasma were collected. The samples were resuspended in RPMI1640 supplemented with 10 wt% FCS, and seeded on the above-mentioned wells whose surface was coated with FCS or complex glycopolymer. After incubation at 37°C for 30 minutes, non-adhered cells were recovered in the form of a cell suspension fluid, and cells adhered to the wells were stained with Pappenheim's stain to identify their type. The ...

Embodiment 3

[0080] Example 3: Primary Separation Through Filtration Separation

[0081] Instead of the density centrifugation method in Example 1, a filter comprising a non-woven polyester fabric with an average pore size of 8 μm was used for primary separation. Maternal blood samples were diluted with biological buffer containing 1 wt% BSA and passed through the filter using natural dropwise addition. Next, pass the buffer only through the filter to wash the red blood cells remaining on the filter. Subsequently, the buffer was passed back through with a syringe pump, and non-adherent cells that did not pass through the filter were recovered. The fraction of cells that did not pass through the filter but did not strongly adhere to the filter was extracted as a primary fraction, which was subjected to secondary fractionation using the carbohydrate-lectin method. Table 4 shows the ratio (fold) of the results of primary separation samples obtained from the above filters to the results of s...

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Abstract

The invention provides a blood cell separation system, which is used for the precise separation and concentration of rare fetal nucleated cells mixed in the blood of pregnant women, so as to facilitate the obtaining of test preparations that can be used for prenatal chromosome / gene diagnosis. The blood cell separation system is characterized in that it includes (1) a primary separation device, which is used to mainly remove non-nucleated red blood cells, white blood cells and platelets from a blood sample taken from a pregnant woman to obtain a primary separation sample, (2) a secondary separation device , for the removal of residual non-nucleated erythrocytes and leukocytes from a primary fraction obtained using the primary fractionation device using the carbohydrate-lectin method to obtain a secondary fraction containing concentrated fetal nucleated cells, and (3) A preparation device for preparing the secondary separation sample obtained by the secondary separation device.

Description

technical field [0001] The present invention relates to a system for separating blood cells using lectins, and more particularly to a system for effectively removing non-nucleated red blood cells and mature white blood cells from a sample containing erythroblasts to separate and concentrate fetal nucleated erythroblasts. The nucleated erythroblasts are fetal nucleated cells present in maternal peripheral blood or umbilical cord blood of pregnant women. It further relates to systems for examining chromosomes and genes. Background technique [0002] In the field of genetic diagnosis, it has long been desired to develop methods for prenatal diagnosis which do not endanger the embryo, ie the fetus. The clinically applied genetic diagnosis methods are invasive devices, such as amniotic fluid diagnosis, chorionic villus sampling and fetal blood collection, especially the fetal cell sampling through amniotic fluid diagnosis will make a definite diagnosis, but it also bears a high ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/07C12N5/073C12N5/078G01N33/537G01N33/569G01N33/68
CPCG01N33/537G01N33/56966G01N33/689G01N2400/00G01N2800/368G01N33/48G01N33/49
Inventor 由良洋文斋藤芳夫北川道弘
Owner NETECH
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