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WO2017030408A1 - Procédé de concentration et de séparation des globules rouges nucléés dans le sang maternel, pour un diagnostic prénatal non invasif - Google Patents

Procédé de concentration et de séparation des globules rouges nucléés dans le sang maternel, pour un diagnostic prénatal non invasif Download PDF

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Publication number
WO2017030408A1
WO2017030408A1 PCT/KR2016/009153 KR2016009153W WO2017030408A1 WO 2017030408 A1 WO2017030408 A1 WO 2017030408A1 KR 2016009153 W KR2016009153 W KR 2016009153W WO 2017030408 A1 WO2017030408 A1 WO 2017030408A1
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Prior art keywords
nucleated
red blood
blood cells
prenatal diagnosis
cells
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English (en)
Korean (ko)
Inventor
한기호
기창석
번영제
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Samsung Life Public Welfare Foundation
Industry Academic Cooperation Foundation of Inje University
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Samsung Life Public Welfare Foundation
Industry Academic Cooperation Foundation of Inje University
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor

Definitions

  • the present invention relates to a method for the concentrated separation of nucleated erythrocytes in maternal blood for non-invasive prenatal diagnosis.
  • diagnostic confirmation of whether a fetus has chromosomal disease is performed by examining embryonic cells to determine karyotype by amniocentesis, chorionic villi or cordocentesis. It can only be obtained by invasive diagnostic tests.
  • fetal cells in the maternal circulation have led many groups to study and develop methods for the isolation and recovery of fetal cells that enable non-invasive prenatal diagnosis.
  • fetal cells that can cross the placental barrier: lymphocytes, trophoblasts, and erythroblasts.
  • lymphocytes lymphocytes
  • trophoblasts trophoblasts
  • erythroblasts erythroblasts.
  • research has focused on the isolation of fetal erythrocytes from peripheral maternal blood and trophoblast cells, which are epithelial cells derived from placenta.
  • fetal cell identification has been made possible by molecular biology methods applied directly to non-cultivated fetal cells.
  • the method is, for example, prenatal Fluorescent In Situ Hybridization (FISH) and Quantitative Fluorescent-Polymerase Chain Reaction (QF-PCR).
  • FISH prenatal Fluorescent In Situ Hybridization
  • QF-PCR Quantitative Fluorescent-Polymerase Chain Reaction
  • NRBCs fetal nucleated erythrocytes
  • the parameters include two morphological features (roundness and morphology of the nucleus) and two characteristics (foetal luminosity and peripheral luminosity) of fetal hemoglobin marking.
  • the protocol uses isolation of mononucleated cells on a density gradient and enrichment by depletion of white blood cells (MACS by antibodies to CD15 and CD45) and gamma anti-hemoglobin antibodies. Separation on a cytofluorimeter by FACS method is defined. Cells identified through multiparameter scores are recovered using a micromanipulator under microscopic observation.
  • MonaLiza Medical Ltd. (US patent 2005/0181429 Al) developed a prenatal genetic analysis method using cervical cervical cells. The method is based on the use of a Pap smear cytobrush for the collection of cervical cervical samples, which samples are processed by cytocentrifugation for the manufacture of slides. Cervical cells are labeled and analyzed under a microscope and their position and coordinates are stored on the slide. The slide is analyzed by FISH and trophoblast cells are identified using the coordinates obtained earlier. A disadvantage of this method is that during processing for the manufacture of the slides, some of the cervical cervical cells are lost and there may be no call due to the absence of trophoblast cells.
  • AVIVA-Biosciences Corporation developed a biochip based enrichment system to separate fetal cells from maternal blood. This method is a high-resolution filtration with most of the red blood cells removed, sorting by a cocktail of high specificity magnetic beads and specific antibodies against fetal antigens, and pores with variable diameters depending on the type of cells to be isolated. Reagents are used that allow for enrichment by a high resolution filtering chamber.
  • the method is limited to the selection of fetal cells by enrichment of the sample, which does not exclude the possibility of risking contaminating maternal cells and consequently unreliable genetic analysis.
  • Nucleus is present in erythroblasts, immature erythrocytes, in the blood of pregnant women, and genetic diagnosis is possible. Since most of the nucleated erythrocytes in the blood of pregnant women are presumed to be caused by the fetus. . In addition, since it is a mononuclear cell, it is very useful for the diagnosis of hereditary diseases, and it exists a lot in the early stages of pregnancy, and in most cases, the blood type with the fetus is suitable, so it can be estimated that the red blood cells of the fetus will not be destroyed even if they cross into the blood of the pregnant woman. Do. In addition, nucleated erythrocytes have a short lifespan, so the results of previous pregnancies do not affect the genetic analysis of the pregnancies.
  • FACS Fluorescent activated cell sorting
  • MCS magnetic activated cell sorting
  • the isolated nucleated red blood cells are used for fluorescent in situ hybridization (hereinafter referred to as FISH) or PCR to determine the sex of the fetus and report the genetic aspects of the fetus.
  • FISH fluorescent in situ hybridization
  • PCR PCR
  • a single cell identified as a fetus under a microscope is obtained by using a micromanipulator, and the cells are amplified by a primer extension preamplification method (hereinafter abbreviated as PEP) using a random primer to amplify the amount of DNA present in a single cell.
  • PEP primer extension preamplification method
  • An object of the present invention is to provide a method for concentrated separation of nucleated red blood cells in maternal blood for a new non-invasive prenatal diagnosis to solve the problems of the prior art as described above.
  • the present invention to solve the above problems
  • a fourth step of separating and removing leukocytes provides a concentrated separation method of nucleated red blood cells in maternal blood for non-invasive prenatal diagnosis comprising a.
  • Figure 1 shows a schematic diagram of the concentrated separation method of nucleated erythrocytes in maternal blood for non-invasive prenatal diagnosis according to the present invention.
  • the method for enriching and separating nucleated erythrocytes in maternal blood for non-invasive prenatal diagnosis selectively concentrates erythrocytes from maternal blood, and then sediments including maternal-derived red blood cells and fetal-derived nucleated red blood cells.
  • a erythrocyte lysis buffer is added to the mass to selectively dissolve and remove the parent-derived non-nucleated erythrocytes, and then the leukocytes are separated by magnetophoresis.
  • hydroxyethyl starch or dextran is added as an erythrocyte sedimentation agent. It is characterized by separating into a concentrate and a first supernatant.
  • hydroxyethyl starch or dextran is added as an erythrocyte sedimentation agent that over-aggregates erythrocytes, erythrocytes They form agglomerates and pile up together.
  • the second step is hydroxyethyl starch or dextran to erythrocyte sedimentation in the first concentrate It is characterized in that it further comprises a 2-1 step of separating into a second concentrate and a second supernatant by adding zero.
  • the third step of dissolving maternal-derived non-nucleated erythrocytes in the concentrated erythrocytes sample is a erythrocyte lysis buffer in the second concentrate. And selectively dissolving only the non-nucleated erythrocytes derived from the mother to obtain a third concentrate containing the nucleated erythrocytes derived from the fetus.
  • the red blood cell lysis buffer used in the present invention is a material that dissolves the aggregated red blood cells in the concentration process, and it is preferable to use ammonium chloride as a material suitable for removing non-nucleated red blood cells, that is, non-nucleated red blood cells derived from the mother.
  • the fourth step is 4--labeling leukocytes contained in the third concentrate treated with the erythrocyte lysis buffer with magnetic nanoparticles. Stage 1; And
  • the leukocytes contained in the third concentrate are immuno-magnetic nanobeads to which anti-CD45 and anti-66b are attached. To be processed.
  • the nanobeads specifically attach to white blood cells contained in the third concentrate. Leukocytes bound to the magnetic beads are removed by a horizontal magnetophoretic microseparator and nucleated red blood cells are collected.
  • step 4-2 it is possible to use an apparatus capable of separating leukocytes by magnetophoresis without limitation.
  • Korean Patent Application No. 2012-0138684 "Method for separating fine particles using magnetophoresis and method for separating fine particles using the same", It is possible to perform steps 4-2.
  • step 4-2 Specifically, in step 4-2
  • a lower substrate comprising a patterned magnetic microstructure
  • the magnetic microstructure is a plurality of linear structures, using a microparticle separation device using a magnetophoresis pattern formed on the lower substrate to have a predetermined inclination angle with respect to the flow direction of the sample in the separation microfluidic channel region Characterized in that.
  • the microfluidic channel is
  • An injection microfluidic channel region including a plurality of injection portions into which a blood sample and a buffer are injected;
  • the width of the discharge portion from which the white blood cells are separated is greater than the width of the discharge portion from which the red blood cells including the nucleated red blood cells are separated.
  • the magnetic microstructure may be made of a ferromagnetic material or an alloy having ferromagnetic properties, and the flow of a sample of the magnetic microstructure It is possible to change the inclination angle with respect to the direction, the thickness of the magnetic microstructures, the spacing between the plurality of magnetic microstructures formed, the number of installation, the size of the external magnetic field source and the flow rate of the fluid in the microfluidic channel.
  • the flow rate in the microfluidic channel is preferably maintained at 500 ⁇ l / h or less.
  • the leukocytes are separated from the discharge portion to be discharged.
  • the ferromagnetic wire is preferably bent 100 ⁇ m at a right angle and formed parallel to the external magnetic field before reaching the wall of the microchannel.
  • the concentrated separation method of nucleated erythrocytes in maternal blood for non-invasive prenatal diagnosis undergoes erythrocyte sedimentation and erythrocyte lysis to dissolve maternal-derived non-nucleated erythrocytes, and separation method by magnetophoresis to separate and remove leukocytes.
  • blood is collected only from the mother, and only nucleated red blood cells derived from the fetus can be separated from the collected blood with high efficiency.
  • 1 is to prepare maternal blood to concentrate red blood cells, lysed aggregated red blood cells using a red blood cell lysis buffer, and then the nucleus in the mother blood for non-invasive prenatal diagnosis according to the present invention comprising the step of separating and removing leukocytes Schematic of the concentrated separation of red blood cells.
  • Figure 2 shows the leukocyte removal and nucleated erythrocyte loss rate in the first supernatant, the second supernatant and the third concentrate prepared in the course of performing red blood cell coagulation reaction and red blood cell lysis in the leukocyte patient blood sample according to the present invention. For the graph.
  • Figure 3 is leukocytes in the first supernatant, the second supernatant and the third concentrate prepared in the process of carrying out the erythrocyte hyperaggregation reaction in the leukocyte patient blood sample and the lysis of red blood cells, which are non-nucleated cells, Nucleated erythrocytes are labeled with fluorescence and the relative percentages of erythrocytes and nucleated erythrocytes measured using a fluorescent activated cell sorter are measured.
  • FIG. 4 is a schematic view and an illustration of a magnetophoretic separation apparatus according to the present invention.
  • Figure 5 is a graph showing the measurement of the number of cells of each white blood cells separated and discharged by varying the flow rate (250, 500, 1000 ⁇ l / h) by the magnetic domain separation apparatus according to the present invention.
  • Figure 6 shows the results of the fetal micronucleated erythrocytes isolated from maternal blood by fluorescent labeling by fluorescence and confirmed by confocal microscopy.
  • peripheral blood 18 samples of peripheral blood were collected from pregnant women 12-27 weeks of gestation.
  • peripheral blood provided from healthy persons and erythrocyte samples provided from three leukemia patients were collected to demonstrate the efficiency of the red blood cell separation method according to the present invention.
  • the second step 200 ⁇ l of HetaSep reagent (STEMCELL technologies) containing hydroxyethyl starch or dextran as red blood cell settler is mixed and incubated at 37 ° C. for 15 minutes to settle the red blood cell pellet. 1 concentrate and the first supernatant containing a plurality of leukocytes formed on the first concentrate. The first supernatant containing a plurality of leukocytes formed on the first concentrate was removed.
  • HetaSep reagent STMCELL technologies
  • the first concentrate containing the red blood cell pellet was resuspended in PBS containing 2% FBS.
  • PBS containing 2% FBS.
  • HetaSep reagent containing hydroxyethyl starch or dextran as an erythrocyte sedimentation agent, the red blood cell coagulation process is performed one more time. The supernatant was separated.
  • the second supernatant was removed, and the second concentrate was treated with BD Pharm LyseTM Lysing buffer (BD Biosciences) for 10 minutes at 4 ° C. After washing twice with PBS containing 2% BSA and suspended again with 50ul PBS containing 0.2% BSA to prepare a third concentrate.
  • BD Pharm LyseTM Lysing buffer BD Biosciences
  • the number of erythrocytes including leukocytes and nucleated erythrocytes in the first supernatant, the second supernatant and the third concentrate prepared from the three leukocyte patient blood samples prepared in Example 1 was determined by a hemocytometer (Marynfeld). And the results are shown in Tables 1 to 3 and FIG. 2 below.
  • the percentage of nucleated erythrocytes in the first supernatant and the second supernatant is 2.03%, compared to 93.98% in the third concentrate prepared using the red blood cell settling agent and the erythrocyte lysis buffer. , 3.99%, it can be seen that the nucleated red blood cells are mostly concentrated in the third concentrate.
  • leukocytes were labeled with anti-CD45-Alexa647 (AbD Serotec) for 30 minutes at 4 ° C. and nucleated erythrocytes were labeled with anti-GPA-FITC (Biolegend) to stain nucleated cells obtained from the samples. Then, using a fluorescent activated cell sorter (FACSCalibur, BD Biosciences) to measure the relative percentage of erythrocytes and nucleated cells (relative percentage) and the results are shown in FIG.
  • FACSCalibur fluorescent activated cell sorter
  • the first supernatant and the second supernatant generated in the separation process according to the present invention contain nucleated red blood cells as 0.03% and 0.16%, respectively, while the third concentrate contains nucleated red blood cells as 47.89. %, And the first supernatant and the second supernatant contain white blood cells as 99.96% and 99.80%, while the third concentrate contains 51.73% as white blood cells.
  • FIG. 4 shows a magnetophoretic separation device manufactured according to an embodiment of the present invention.
  • the magnetophoretic separation device manufactured according to an embodiment of the present invention includes two inlets, two outlets, and a ferromagnetic wire array of Ni 0.8 Fe 0.2.
  • the ferromagnetic wire array is placed on the lower substrate and is formed to form an angle of 5.7 ° with the direction in which the fluid flows.
  • Microchannels are manufactured to a height of 50 ⁇ m and a width of 1,000 ⁇ m. The width of the sample and the buffer inlet is 500 ⁇ m.
  • the ferromagnetic wire When an external magnetic field is applied, the ferromagnetic wire produces a high magnetic field.
  • the generated magnetic field (Fm) moves the white blood cells labeled with magnetic beads. That is, if the magnetic force Fm and the hydrodynamic drag force (Fd) are compared and the magnetic force is sufficient to move the white blood cells, the white blood cells labeled with magnetic beads move horizontally along the ferromagnetic wire and are discharged through outlet 2.
  • nucleated red blood cells are collected and moved to outlet 1 without being affected by magnetic beads. There is a non-specific binding between the nucleated red blood cells and the magnetic beads, but a few nucleated red blood cells that can be affected by the magnetic force may be lost.
  • the widths of the outlets 1 and 2 are different, and the widths are 800 ⁇ m and 200 ⁇ m, respectively.
  • the ferromagnetic wire was bent 100 mu m at almost right angles and formed parallel to the external magnetic field before reaching the wall of the microchannel.
  • the number of leukocytes separated and discharged was measured while adjusting the flow rate to 250, 500, and 1000 ⁇ l / h using a blood sample of a normal person. The results are shown in FIG.
  • Fetal-derived DNA in maternal plasma's total cell-free DNA is typically about 11% (Xu. Et al., 2013).
  • studies have shown that the number of fetal-derived nucleated cells in 1 ml of maternal blood is one or more (Kwon et al., 2007, Sekizawa et al., 2007, Mavrou et al., 2007). Therefore, the number of leukocytes incorporated per 1 ml of mother's blood is less than 10 because the purity of nucleated cells is higher in fetal-derived cell-free fetal DNA compared to mother's cell-free DNA. Should be.
  • a third concentrate was prepared by performing red blood cell concentration, lysis, and separation processes of the first to third stages in the same manner as in Example 1 with respect to 18 ml of mother's blood.
  • Example 2-1 In order to label the leukocytes contained in the concentrate prepared in Example 2-1, after mixing together with anti-CD45 and anti-CD66b antibodies, magnetic nanobeads (STEMCELL Technologies Inc.) were mixed and mixed at room temperature. Incubate for 120 and 80 minutes respectively.
  • nucleated erythrocytes were separated using a separation device prepared in the above preparation.
  • nucleated erythrocytes and white blood cell numbers isolated from 18 maternal blood samples were measured. As shown in FIG. 7, the nucleated red blood cells in the final concentrate separated by the nucleated red blood cell concentration and separation method according to the present invention have 1 to 396 nucleated red blood cells, whereas 0 to 6 leukocytes have high efficiency in the nucleated red blood cells according to the present invention. It was confirmed that the concentration and separation as.
  • the cells discharged to outlet 1 of the horizontal self-phoretic microseparator were collected in a 1.5 ml tube, and the separated leukocytes and red blood cells were membrane-permeable nucleic acid fluorescent dyes (Hechest; 34580), each cell nuclei were stained, white blood cells were stained with anti-CD45-Alexa647, and nucleated erythrocytes were stained with anti-GPA-FITC. Stained cells were confirmed by confocal microscopy (LSM 510 META; Carl Zeiss) and the results are shown in FIG. 6.
  • LSM 510 META Carl Zeiss
  • the isolated nucleated erythrocytes had a round nucleus in the cell, not stained with the leukocyte label, it was confirmed that the stained with the nucleated cell label was confirmed that the nucleated red blood cells were isolated by the present invention.
  • the Y-chromosome specific gene (SRY gene) was analyzed.
  • genomic DNA (gDNA) in the concentrated sample was subjected to WGA and diluted 100-fold with RNAase-free distilled water (REPLI-g Single Cell Kit; Qiagen).
  • PCR was performed using AccuPower Hotstart PCR PreMix, a PCR mix powder from BIONEER. 20 ⁇ l of the PCR mix, 4 ⁇ l of the forward and reverse primers, 1 ⁇ l of the template gDNA, and 12 ⁇ l of distilled water were mixed in sequence and 94 ° C. 30 seconds, 55 ° C. 30 seconds, using a Gene Amp PCR System 9700 from Applied Biosystems, The reaction was carried out 30 times at 72 ° C for 30 seconds and finally extended at 72 ° C for 10 minutes.
  • SRY gene amplification results were detected in the nucleated erythrocytes isolated after concentration, confirming that the nucleated erythrocytes according to the present invention are derived from the fetus.
  • the concentrated separation method of nucleated erythrocytes in maternal blood for non-invasive prenatal diagnosis undergoes erythrocyte sedimentation and erythrocyte lysis to dissolve maternal-derived non-nucleated erythrocytes, and separation method by magnetophoresis to separate and remove leukocytes.
  • blood is collected only from the mother, and only nucleated red blood cells derived from the fetus can be separated from the collected blood with high efficiency.

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Abstract

La présente invention concerne un procédé permettant de concentrer et de séparer des globules rouges nucléés maternels, pour un diagnostic prénatal non invasif. Selon la présente invention, le procédé permettant de concentrer et de séparer des globules rouges nucléés dans le sang maternel, pour un diagnostic prénatal non invasif, utilise de manière séquentielle un procédé d'hyperagrégation de globules rouges, un procédé de lyse de globules rouges pour lyser des globules rouges de cellules non nucléées dérivées de la mère, et une leucaphérèse utilisant la magnétophorèse, permettant ainsi, contrairement à un procédé classique, de ne collecter que le sang provenant de la mère, et de ne séparer que les globules rouges nucléés dérivés du fœtus, avec une efficacité élevée, à partir du sang maternel collecté.
PCT/KR2016/009153 2015-08-19 2016-08-19 Procédé de concentration et de séparation des globules rouges nucléés dans le sang maternel, pour un diagnostic prénatal non invasif Ceased WO2017030408A1 (fr)

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CN106969964A (zh) * 2017-02-23 2017-07-21 宁波美晶医疗技术有限公司 一种基于微流控及免疫磁分离的血液中稀有细胞的负相富集方法及试剂盒
CN109735430A (zh) * 2019-01-28 2019-05-10 武汉纺织大学 一种三维磁泳分离的微流控芯片
WO2023275880A1 (fr) * 2021-06-30 2023-01-05 Regrow Biosciences Private Limited Procédé d'obtention d'une population de csh, d'une population de lymphocytes t et d'une population de cellules nk et compositions associées
WO2023009782A1 (fr) * 2021-07-30 2023-02-02 General Electric Company Techniques d'isolation ou d'analyse des pathogènes bactériens à partir d'échantillons prélevés sur des patients

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KR102048616B1 (ko) * 2017-12-28 2019-11-25 주식회사 지노바이오 분리칩
KR102048617B1 (ko) * 2017-12-28 2020-01-08 주식회사 지노바이오 분리칩
KR102826451B1 (ko) * 2023-09-01 2025-06-27 사회복지법인 삼성생명공익재단 신생아로부터 비침습적으로 분리된 검체를 이용한 신생아의 검체 분석 방법

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106969964A (zh) * 2017-02-23 2017-07-21 宁波美晶医疗技术有限公司 一种基于微流控及免疫磁分离的血液中稀有细胞的负相富集方法及试剂盒
CN106969964B (zh) * 2017-02-23 2020-02-18 宁波美晶医疗技术有限公司 一种基于微流控及免疫磁分离的血液中稀有细胞的负相富集方法及试剂盒
CN109735430A (zh) * 2019-01-28 2019-05-10 武汉纺织大学 一种三维磁泳分离的微流控芯片
WO2023275880A1 (fr) * 2021-06-30 2023-01-05 Regrow Biosciences Private Limited Procédé d'obtention d'une population de csh, d'une population de lymphocytes t et d'une population de cellules nk et compositions associées
WO2023009782A1 (fr) * 2021-07-30 2023-02-02 General Electric Company Techniques d'isolation ou d'analyse des pathogènes bactériens à partir d'échantillons prélevés sur des patients

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