WO2017057076A1

WO2017057076A1 - Method for obtaining nucleated erythrocytes and method for identifying nucleated erythrocytes

Info

Publication number: WO2017057076A1
Application number: PCT/JP2016/077555
Authority: WO
Inventors: 中津　雅治
Original assignee: 富士フイルム株式会社
Priority date: 2015-09-28
Filing date: 2016-09-16
Publication date: 2017-04-06

Abstract

Provided are a method for obtaining nucleated erythrocytes and a method for identifying nucleated erythrocytes allowing for reliable analysis of targeted cells. The method for obtaining nucleated erythrocytes comprises: a fixing step of subjecting maternal blood taken from a pregnant mother to a cell fixation process; a concentration step of concentrating the maternal blood after the fixing step; and an identification step of identifying the nucleated erythrocytes in maternal blood, which was concentrated by the concentration step. The method for identifying nucleated erythrocytes comprises an analysis step of genetically analyzing the nucleated erythrocytes, and all of the steps are handled in a solution.

Description

Nucleated red blood cell acquisition method and nucleated red blood cell identification method

The present invention relates to a method for obtaining nucleated red blood cells and a method for identifying nucleated red blood cells, and more particularly to a method for obtaining nucleated red blood cells and a method for identifying nucleated red blood cells for examining nucleated red blood cells in pregnant maternal blood.

As a prenatal diagnosis, amniocentesis, villus examination, umbilical cord puncture, and the like have been conventionally performed.

On the other hand, it is known that fetal cells migrate into the blood of a pregnant mother (hereinafter also simply referred to as “maternal”), and these fetal cells circulate in the mother together with blood. Thus, there is a demand for using maternal blood to reliably analyze fetal cell chromosome DNA (deoxyribonucleic acid) in maternal blood with high reproducibility.

However, it is said that there are only a few fetal cells (nucleated erythrocytes) present in maternal blood in a few milliliters of maternal blood. This is a major issue in conducting prenatal diagnosis using blood.

As a method of obtaining nucleated red blood cells that are fetal cells from maternal blood, there is a method of concentrating nucleated red blood cells, for example, a technique of removing plasma components and unnecessary red blood cell components using density gradient centrifugation. In addition, using an antibody that specifically immunoreacts on the surface or inside of a blood cell, a technique for separating blood cells by fluorescence or magnetism labeled with the antibody (FACS method: Fluorescence Activated Cell Sorting, MACS method: Magnetic Activated Cell Sorting Etc.), nucleated red blood cells, which are fetal cells in maternal blood, are obtained by combining the method of utilizing the difference in resistance to hemolysis or pH between maternal red blood cells and fetal red blood cells, alone or in combination. Yes.

These methods are useful techniques for increasing the concentration of fetal nucleated red blood cells in the blood and increasing the probability of obtaining the target cells. However, although these techniques can increase the probability of the target cell, they are not sufficient for reliably obtaining fetal nucleated red blood cells in a short time.

Also, cell fixation processing is performed to stabilize cell morphology and tissue structure. For example, in Patent Document 1 below, as a method for obtaining cells for identifying fetal genetic characteristics, cell fixation is performed by concentrating cells and then suspending them in paraformaldehyde. It is also described that it may be performed in a suspended state on a slide glass. Patent Document 2 describes that as a method for preparing a sample of cells having micronuclei, it can be maintained in a stable state by being suspended in glutaraldehyde and can be made into a non-slide sample. Patent Document 3 discloses a method for concentrating nucleated red blood cells as a method for obtaining nucleated red blood cells, which are fetal cells in maternal blood, for example, plasma components and maternal red blood cells using density gradient centrifugation. Technology that removes components, antibody that specifically immunoreacts with proteins on the surface of leukocytes, magnetic separation of maternal leukocytes (MACS method), antibodies that specifically immunoreact with the gamma chain of fetal hemoglobin It describes that cells are fixed after concentration using a technique (FACS method) or the like that separates fetal nucleated red blood cells using a fluorescent dye.

Japanese National Patent Publication No. 7-509136 JP 2001-149098 A Special table 2010-525832 gazette

However, in any of the methods described in the above-mentioned patent documents, in the process of concentrating cells when obtaining target cells, cell aggregation or cell disruption by centrifugation, pipetting, etc. In addition, cell loss due to cell attachment to the microtube wall occurred. When searching for extremely rare cells such as fetal cells in maternal blood, it is necessary to avoid losing the target cells as much as possible. Moreover, although DNA amplification is performed after acquiring the target cell, it is necessary to perform DNA amplification reliably.

The present invention has been made in view of such circumstances, and a method for obtaining nucleated red blood cells and a method for preventing the loss of cells when concentrating cells and capable of reliably analyzing the target cells. An object is to provide a method for identifying nuclear red blood cells.

In order to achieve the above object, the present invention provides a fixing step for cell-fixing maternal blood collected from a pregnant mother, a concentration step for concentrating the maternal blood after the fixing step, and maternal blood concentrated by the concentration step. And a method for identifying the nucleated red blood cells.

According to the method for obtaining nucleated red blood cells of the present invention, since the maternal blood is concentrated after the cell fixing treatment, cell aggregation, cell disruption, centrifugation by operations such as centrifugation and pipetting in the concentration step are performed. Cell loss due to cell adhesion to the wall of the tube or the wall of the microtube can be prevented. Therefore, target nucleated red blood cells can be stably obtained.

In another aspect of the present invention, it is preferable that the cell fixing treatment solution used in the fixing step contains at least one of a glutaraldehyde solution and a paraformaldehyde solution.

According to this aspect, by using a cell fixing treatment solution containing at least one of a glutaraldehyde solution and a paraformaldehyde solution, it is possible to fix cells without agglutinating blood cells. In addition, resistance to various stresses on blood cells in the operation of concentration treatment in the concentration step can be imparted to blood cells. Therefore, cell loss during the concentration step can be prevented, and target nucleated red blood cells can be reliably obtained.

In another aspect of the present invention, the concentration step is preferably performed by density gradient centrifugation.

According to this aspect, by performing the concentration step by density gradient centrifugation, the concentration of nucleated red blood cells in the maternal blood can be increased efficiently.

In another embodiment of the present invention, it is preferable to obtain a fraction enriched with nucleated red blood cells by cell sorting using an antigen-antibody reaction after concentration by density gradient centrifugation.

According to this aspect, nucleated red blood cells can be further concentrated efficiently by performing cell sorting using antigen-antibody reaction after concentration by density gradient centrifugation.

In another aspect of the present invention, the identification step preferably includes a step of identifying fetal nucleated red blood cells, and is performed by identifying an antibody that specifically reacts with fetal cells.

According to this aspect, fetal-derived nucleated red blood cells can be identified in the identifying step by performing the identifying step using an antibody that specifically reacts with fetal-derived cells.

In another aspect of the present invention, the method has a washing step of washing maternal blood with a washing solution containing the first cell treatment agent after at least one of the fixing step and the concentration step, The cell treatment agent is preferably at least one selected from albumin, polyethylene glycol, polyvinyl alcohol, and polyvinyl pyrrolidone.

According to this aspect, the cytoplasm is prevented from being destroyed during each treatment operation by washing with the washing solution containing the first cell treatment agent either after the fixing step or after the concentration step. Can do. Therefore, target nucleated red blood cells can be stably obtained. As the first cell treatment agent, the above-mentioned cell treatment agent can be preferably used.

In another aspect of the present invention, the washing solution contains a second cell treatment agent, and the second cell treatment agent is at least one selected from sucrose, melezitose, trehalose, and arginine. preferable.

According to this aspect, the second cell treatment agent can be contained in the blood cells by including the second cell treatment agent in the washing liquid. Therefore, even if the water content of the maternal blood decreases during the treatment operation, the blood cells contain the second cell treatment agent, so that physical damage can be reduced. Furthermore, in the analysis after cell isolation, inhibition of DNA amplification can be reduced, and gene analysis can be reliably performed. As the second cell treatment agent, the above-mentioned cell treatment agent can be preferably used.

In another aspect of the present invention, it is preferable to have an acquisition step of collecting the nucleated red blood cells identified in the identification step.

According to this aspect, by collecting the nucleated red blood cells identified in the identification step, operations such as gene analysis can be easily performed.

In order to achieve the above object, the present invention has an analysis process for genetic analysis of nucleated red blood cells obtained by the method for obtaining nucleated red blood cells described above, and in all steps from the fixing process to the analysis process, A method for identifying nucleated red blood cells handled in a solution is provided.

According to the method for identifying nucleated red blood cells of the present invention, blood cells undergo a dry state by handling the cells in a solution in all the steps up to the analysis step for genetic analysis of maternal blood collected from a pregnant mother. And genetic analysis can be performed. Therefore, since physical damage of blood cells can be reduced, gene analysis can be reliably performed.

According to the method for obtaining nucleated red blood cells and the method for identifying nucleated red blood cells of the present invention, after performing the fixing step of fixing the maternal blood to the cells, the concentration step is performed, so that the enucleation and the cells in the concentration step are performed. There is no need for stress on cells due to aggregation, cell disruption, pipetting, centrifugation, etc., cell loss due to cell adhesion to the wall of a centrifuge tube or a microtube tube, and its concerns, and blood processing operations can be performed with confidence. In addition, nucleated red blood cells including extremely rare fetal nucleated erythrocytes can be reliably obtained from the maternal blood, and as a result, the acquisition rate of fetal nucleated red blood cells can be increased.

FIG. 1 is a flowchart showing a procedure of a method for identifying nucleated red blood cells.

Hereinafter, the method for obtaining nucleated red blood cells and the method for identifying nucleated red blood cells of the present invention will be described with reference to the accompanying drawings. In the present specification, “to” is used to mean that the numerical values described before and after it are included as a lower limit value and an upper limit value.

FIG. 1 is a flowchart showing a procedure of a method for identifying nucleated red blood cells. The identification method of nucleated red blood cells includes a preparation step (step S12) for collecting maternal blood from a pregnant mother, a fixing step (step S14) for performing cell fixation processing on the maternal blood collected in the preparation step, and a mother body after the fixing step. A concentration step (step S16) for concentrating blood, an identification step for identifying nucleated red blood cells in the maternal blood after the concentration step (step S18), and an acquisition step (step for collecting the nucleated red blood cells identified in the identification step) S20) and an analysis step (step S22) for genetic analysis of the nucleated red blood cells acquired in the acquisition step. The method for obtaining nucleated red blood cells according to the present embodiment includes three steps from the fixing step (step S14) to the identifying step (step S18). The method for identifying nucleated red blood cells includes the fixing step (step S14) to the analyzing step (step S14). It consists of five steps of step S22). Hereinafter, each step will be described.

<Preparation process (step S12)>
The preparation step is a step of collecting maternal blood from a pregnant mother and preparing the mother blood as a sample for cell fixation treatment.

The maternal blood is preferably peripheral blood of a pregnant mother with low invasiveness. Maternal peripheral blood includes maternal eosinophils, neutrophils, basophils, monocytes, lymphocytes and other white blood cells, nucleated red blood cells, maternal nucleated red blood cells, and fetal origin Nucleated red blood cells and the like. Fetal nucleated red blood cells are said to be present in maternal blood from about 6 weeks after pregnancy. In this embodiment in which prenatal diagnosis is performed, the peripheral blood of the mother is examined after about 6 weeks after pregnancy. Examples of cells having a nucleus in the peripheral blood include maternally derived white blood cells, nucleated red blood cells, and rare fetal-derived nucleated red blood cells.

Fetal nucleated red blood cells are red blood cell precursors that pass through the placenta and are present in the maternal blood. Since the erythrocytes have chromosomes, fetal chromosomes and fetal genes can be obtained by means of low invasiveness. The fetal nucleated red blood cells are said to be present in a ratio of about 1 in 10 ⁶ cells in the maternal blood, and the existence probability is very low in the peripheral blood of the maternal blood.

When collecting maternal blood from a pregnant mother, the pregnant maternal blood can be collected in a blood collection tube containing an anticoagulant. The collected maternal blood can be diluted to about 1.5 to 10 times with physiological saline or phosphate buffered saline (PBS). In addition, staining dyes and antibodies can be added to and retained in maternal blood (including diluted ones), or pH can be adjusted. In this embodiment, the concentration step is performed after the fixing step, thereby preventing cell alteration during the concentration step and minimizing changes in morphological characteristics. Do not perform blood cell concentration (separation) operations (before the fixation step).

<Fixing step (Step S14)>
Next, in the fixing step, maternal blood cell fixing processing is performed. Cell fixation treatment is generally performed for the purpose of stabilizing cell morphology and tissue structure, strengthening a cell sample, improving staining properties, inactivating proteolytic enzymes, contamination of microorganisms, and inhibiting corrosion. It is possible to coagulate and dehydrate the cytoplasm and proteins in the nucleus to prevent cell alteration and minimize changes in morphological characteristics. The fixing step of the present embodiment is performed mainly for the purpose of stopping the differentiation of erythroid cells including nucleated red blood cells and strengthening the cell structure.

Examples of immobilization methods include chemical immobilization for molecular cross-linking and protein insolubilization, and physical immobilization for drying and freezing. After perfusion with physiological saline, perfusion method to quickly fix in deep tissue by perfusing fixative, soak in cryogenic freezing embedding agent (OCT (OptimalCutting Temperature) compound etc.) and freeze A freezing method in which the sample is stored in liquid nitrogen, a drying method in which a sample is air-dried and then heated and fixed on a substrate with a flame or the like can be used.

In this embodiment, any fixing method can be used, but it is necessary to simply fix the cells without aggregating blood cells in the solution. In this case, it is preferable to use a glutaraldehyde solution or a paraformaldehyde solution as the cell fixing treatment solution. As will be described later, in this embodiment, each step up to the analysis step is preferably performed in a solution in order to reduce physical damage of blood cells, and the fixing step is performed by an immersion method or a perfusion method. Preferably it is done.

The cell fixing treatment solution used in the immersion method or perfusion method preferably contains at least one of a glutaraldehyde solution and a paraformaldehyde solution. The concentration of the cell fixing treatment agent is preferably 0.0001% to 25% by weight with respect to the weight of the cell fixing treatment solution (physiological saline or phosphate buffered saline solvent). More preferably, they are 0.001% or more and 10% or less, More preferably, they are 0.01% or more and 6% or less.

The treatment time for the cell fixation treatment can be 5 minutes or longer, but can be set as appropriate according to the concentration of the cell fixation treatment solution. The cell fixing treatment temperature is preferably 1 ° C. or more and 25 ° C. or less, more preferably 2 ° C. or more and 15 ° C. or less, and further preferably 3 ° C. or more and 10 ° C. or less.

<Concentration step (step S16)>
Next, in the concentration step, nucleated red blood cells in the maternal blood subjected to cell fixation treatment in the fixing step are concentrated. Since there are very few nucleated red blood cells contained in maternal blood, it is necessary to concentrate the maternal blood to increase the abundance ratio of nucleated red blood cells. In the present embodiment, by performing a fixing step before the concentration step, stress in the cell due to enucleation, cell aggregation, cell disruption, pipetting, centrifugation, etc., the tube or the tube wall of the microtube in the concentration step Therefore, a fraction containing more nucleated red blood cells can be obtained with certainty.

In the concentration step, methods used for concentrating maternal blood include known methods such as density gradient centrifugation, cell sorting using antigen-antibody reaction (for example, FACS method and MACS method), lectin method, Alternatively, a filter filtration method or the like can be used. In the present embodiment, it is preferable to perform concentration treatment by density gradient centrifugation as a simple concentration method utilizing the characteristics of blood cells.

[Density gradient centrifugation]
The density gradient centrifugation method will be described. The density gradient centrifugation method is a method of separating using the difference in density of components in blood. Density gradient centrifugation is a method that does not use a separation medium, a method that uses one type of separation medium to separate the top and bottom of the separation medium, or uses two types of separation media. Collecting target components (nucleated erythrocytes including fetal nucleated erythrocytes in this embodiment) using a method of separating the density region of the target component so as to be sandwiched between separation media. Can do. Then, nucleated red blood cells can be concentrated from the maternal blood by collecting a fraction containing the target component.

As a method that does not use a separation medium, the blood sample, maternal blood (which may be diluted), is filled into a centrifuge tube and centrifuged, and then the target component is collected to collect nucleated red blood cells. Concentration can be performed.

As a method of using one kind of separation medium, a separation medium is injected into the bottom of a centrifuge tube (or a centrifuge tube), and maternal blood (which may be diluted) is a blood sample on the separation medium. Centrifugation is carried out after laminating the nucleated cells, and nucleated red blood cells can be concentrated by collecting the upper part of the separation medium after centrifugation (which may include a part of the separation medium).

In the method using two types of separation media, the first separation medium is injected into the bottom of the centrifuge tube, the second separation medium is laminated on the first separation medium, and the second separation medium is then separated. The maternal blood (which may be diluted) is laminated on the medium for centrifugation, and then centrifuged, and the layer between the first separation medium and the second separation medium after the centrifugation (first layer) The nucleated red blood cells can be concentrated by collecting a part of one separation medium and / or a part of the second separation medium). In addition, when the centrifuge tube in which the first separation medium is stacked is cooled before the second separation medium is stacked, mixing in the boundary region between the first and second separation media can be suppressed.

International publication WO2012 / 023298 describes the density of maternal blood including fetal nucleated red blood cells. According to the description, the density of nucleated red blood cells derived from fetuses is about 1.065 to 1.095 g / mL, and the density of maternal blood cells is about 1.070 to 1.120 g / mL for red blood cells. Acidocytes are about 1.090 to 1.110 g / mL, neutrophils are about 1.075 to 1.100 g / mL, basophils are about 1.070 to 1.080 g / mL, and lymphocytes are about 1.060. About 1.080 g / mL and monocytes are about 1.060-1.070 g / mL.

The density of the separation medium to be stacked is set to separate fetal nucleated red blood cells having a density of about 1.060 to 1.100 g / mL from other blood cell components in the mother body. For example, in the method using two types of separation media, the density of the center of fetal nucleated red blood cells is about 1.080 g / mL. Therefore, two different separation media (1 0.080 g / mL density separation medium and 1.080 g / mL density separation medium), and adjacent layers are stacked to collect nucleated red blood cells derived from the desired fetus at the interface It becomes possible. Preferably, the density of the first separation medium is set to 1.075 g / mL or more and 1.105 g / mL or less, and the density of the second separation medium is set to 1.025 g / mL or more and 1.075 g / mL or less. Is preferred. More preferably, the density of the first separation medium is 1.080 g / mL or more and 1.100 g / mL or less, and the density of the second separation medium is 1.030 g / mL or more and 1.075 g / mL or less. In the present embodiment, the first separation medium and the second separation medium may be the same type or different types as long as the effect of the present invention can be realized.

Separation media used for density gradient centrifugation include Histopaque (registered trademark) which is a solution containing polysucrose and sodium diatrizoate, OptiPrep (registered trademark) which is a solution containing iodixanol, and a diameter of 15 to 30 nm coated with polyvinylpyrrolidone. A separation medium such as Percoll (registered trademark), which is a solution containing silica sol, and Ficoll-Paque (registered trademark), which is a neutral hydrophilic polymer solution rich in side chains made from sucrose, may be used. it can. In the present embodiment, Histopaque or OptiPrep and a mixture thereof can be preferably used.

[Method using antigen-antibody reaction]
Next, a method using an antigen-antibody reaction will be described. The method using the antigen-antibody reaction may be performed alone, but in this embodiment, the antigen-antibody reaction is performed after concentration treatment by density gradient centrifugation in combination with concentration treatment by density gradient centrifugation. It is preferable to carry out a method using

The method using the antigen-antibody reaction is a method in which components on the blood cell surface or inside are used as antigens to react with antibodies and used for separation and concentration of blood cells. For example, anti-CD45 antibody for leukocytes, anti-CD235a antibody for erythrocytes, anti-CD71 antibody for young blood cells, antibodies that specifically react with γ or ε chains of fetal hemoglobin, anti-i antibodies that react with fetal blood cells, etc. Used to separate and concentrate blood cells by detecting fluorescent dye (FACS), magnetic substance (MACS), polymer beads or the like labeled on the antibody.

The antigen-antibody reaction is preferably performed before the immobilization treatment when a blood cell surface antigen is used. In this case, the antigen-antibody reaction can be performed in a preparation step (step S12). However, in the present invention, since the concentration step (step S16) is performed after the fixing step (step S14), the concentration operation using the antigen-antibody reaction is performed after the fixing step.

In addition, when using an antigen inside a blood cell, a cell membrane permeation treatment or the like may be required to promote the entry of the labeled antibody into the cell. In this case, it can be carried out before the concentration treatment operation. A known technique using a cell membrane permeabilizing agent such as Triton (registered trademark) X-100 or NP-40 can be used for the cell membrane permeabilizing treatment.

As an antibody to be used, it can be used by appropriately selecting from known antibodies. For example, many antibody reagents are sold on behalf of BD Biosciences.

Fluorescent dyes labeled on antibodies can be optimally combined using known techniques depending on the relative fluorescence intensity, antigen density, detection system or number (type) of labels. For example, a large number of fluorescent reagents are sold on behalf of Molecular®Probes (registered trademark).

<Washing process>
In the present embodiment, it is preferable to have a cleaning process for cleaning maternal blood. The washing process can be performed in any of the preparation process (step S12) to the identification process (step S18), but is preferably performed after the fixing process, after the concentration process, or after specimen preparation. Moreover, a washing | cleaning process is not limited to 1 time, It can also carry out after each process. By performing after the fixing step, the fixing processing solution can be removed.

When washing maternal blood, use phosphate buffer (PBS: Phosphate Buffered Saline) as the washing solution, add the washing solution to the maternal blood or maternal blood fraction, disperse the blood cells, and then centrifuge It can be washed.

In the present embodiment, the first cell treatment agent is contained in the washing solution, and the maternal blood can be washed with the washing solution containing the first cell treatment agent.

Since the cytoplasm can be protected by washing with a washing solution containing the first cell treatment agent, the occurrence of a nuclear shadow can be reduced when a blood cell smear sample is prepared. Nucleus is a number of traces in the blood cell smear that the contents of the collapsed cells appear to have spread to the ground, and the nucleated blood cells are broken in the blood cell smear and the nucleus is attached to the ground. it is conceivable that. The nuclear shadow causes contamination in cell isolation, and causes variation in the results of genetic analysis after isolation. By washing with a washing liquid containing the first cell treatment agent, cells can be protected and the occurrence of nuclear shadows in smears can be reduced.

As the first cell treatment agent, at least one selected from albumin, polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP) can be used. It is preferable to use albumin, and bovine serum albumin (BSA: Bovine Serum Albumin) is more preferred.

The concentration of the first cell treatment agent in the washing liquid is preferably 1.5% by mass or more and 9.0% by mass or less, and more preferably 2.0% by mass or more and 5.0% by mass or less. . When the concentration is 1.5% by mass or more, the nuclear shadow can be almost eliminated. Moreover, it is preferable to set it to 9.0 mass% or less because the content of the first cell treatment agent does not become an excessive amount while having the effect of eliminating the nuclear shadow. In addition, when using a several component as a 1st cell treatment agent in a washing | cleaning liquid, it is preferable to make the total density | concentration of a component into the said range.

Also, the washing treatment can be performed by containing the second cell treatment agent in the washing solution. As the second cell treatment agent, at least one selected from sucrose, melezitose, trehalose, and arginine is preferably used, and trehalose is particularly preferable. By treating with the second cell treatment agent, when the identification step is performed by preparing a smear, the DNA isolated from the smear can be reliably amplified, and the analysis can be performed reliably. Can do. When the cells smeared on the substrate are dried, the aqueous solution containing the second cell treatment agent penetrates into the cells and is dried in a state where the second cell treatment agent is present in the cells. If drying proceeds in a state where the second cell treatment agent is not present, DNA may be damaged by the drying. However, when the second cell treatment agent is present, the second cell is washed with water. It is assumed that damage to DNA due to drying can be reduced.

The concentration of the second cell treatment agent in the washing liquid is preferably 1.2% by mass or more and 9.0% by mass or less, and more preferably 2.0% by mass or more and 5.0% by mass or less. . By setting the content concentration of the second cell treatment agent to 1.2% by mass or more, DNA amplification can be ensured. Moreover, it is preferable to set it to 9.0 mass% or less because the content of the second cell treatment agent does not become excessive while having the effect of ensuring DNA amplification. In addition, when using as a 2nd cell treatment agent including a some component in a washing | cleaning liquid, it is preferable to make the total density | concentration of a component into the said density | concentration range.

<Identification process (step S18)>
Next, the nucleated red blood cells are identified in the maternal blood fraction in which the nucleated red blood cells are concentrated by the concentration step. The identification step preferably includes identification of the presence or absence of stained or labeled cell nuclei, identification of fetal or maternal cells, and identification of erythrocytes by observation of hemoglobin spectral information or blood cell morphology. Identification of cell nuclei, fetal or maternal cells, and erythrocytes can be performed by fluorescence observation, bright field observation, or a combination thereof. This identifies nucleated red blood cell candidates that are likely to be of fetal origin. Here, the red blood cell system means red blood cells or nucleated red blood cells.

[Sample preparation]
In the present embodiment, it is preferable to handle maternal blood in a solution in all steps from the collection of maternal blood from the pregnant mother to the analysis step of performing genetic analysis. By handling maternal blood in solution, blood cells can perform genetic analysis without going through a dry state, so physical damage to blood cells can be reduced. it can.

When the identification step is performed in a solution, the fraction enriched with nucleated red blood cells after the concentration step can be fractionated into a microwell plate or a microtube.

Also, a maternal blood fraction enriched with nucleated red blood cells obtained in the concentration step can be smeared on a substrate to produce a smear, and the identification step can be performed. As a method for preparing a smear, it can be performed by a known technique.

Further, in the washing step, when the second cell treatment agent is not included in the washing solution, the treatment is performed by bringing a solution containing the second cell treatment agent into contact with blood cells of maternal blood at the time of preparing a smear. be able to. Moreover, you may perform the process by a 2nd cell processing agent in both the washing | cleaning process and the time of preparation of a smear.

The treatment of the second cell treatment agent in smearing can be used in addition to the maternal blood fraction immediately before smearing the concentrated maternal blood fraction on the substrate. Moreover, it can carry out by making the solution containing a 2nd cell processing agent contact the smear sample by which the fraction of the concentrated maternal blood was smeared on the board | substrate.

When the second cell treatment agent is added to the maternal blood fraction before smearing, the concentration of the second cell treatment agent in the maternal blood fraction (or in the dilution of the fraction) is 1.2. It is preferable to add so that it may become mass% or more and 9.0 mass% or less, More preferably, it is 2.0 mass% or more and 5.0 mass% or less.

Further, when the substrate on which the maternal blood fraction is smeared is brought into contact with the solution containing the second cell treatment agent, the concentration of the second cell treatment agent is 1.2 mass% or more and 9.0 mass% or less. It can be processed by contacting with a method of loading a buffer such as a certain phosphate buffer or a method of immersing in a buffer.

After the smearing of the maternal blood fraction on the substrate, the smear is dried. The smear can be dried by air drying or the like. When the second cell treatment agent is present in the blood cells at the time of drying, damage to the cells due to the drying can be reduced. Since the second cell treatment agent only needs to be present when the smear is air-dried, it can be brought into contact with blood cells either in the washing step and before or after smearing the fraction on the substrate. Good.

Next, the identification of cell nuclei, the identification of fetal cells, and the identification of erythrocytes will be described.

[Identification of cell nucleus]
For the identification of the presence or absence of cell nuclei, staining of cell nuclei with a staining dye can be used. Staining is to stain cells using dyes or pigments so that cells in maternal blood, particularly cells having cell nuclei, can be identified by light information.

In this embodiment, a fluorescent dye or a general dye can be used as the nuclear dye.

Fluorescent dyes are dyes that emit light by absorbing irradiated light energy, and are often used for cell staining. In the present embodiment, the following nuclear staining fluorescent dye can be appropriately selected and used. 7-AAD (7-amino-actinomycin D), ACMA (9-Amino-6-chloro-2-methoxyacridine), Acridine homodimer, Acridine orange, Actinomycin D, BCECF-AM, CyTRAK Orange, DAPI (4 ', 6- diamidino-2-phenylindole), Dihydroethidium, DRAQ5, DRAQ7, Ethidium bromide, Ethidium homodimer-1 (EthD-1), Ethidium homodimer-2 (EthD-2), Ethidium monoazide, Ethidium Monoazide Bromide (ech), (Registered trademark) 33258 (bis-benzimide), Hoechst 33342, Hoechst 34580, Hydroxystilbamidine, LDS 751, NeuroTrace (registered trademark), Nile blue, Nuclear yellow, NucRedR Live 647, Propidium iodide (PI), SYBR (registered trademark) Green , SYTO (registered trademark), SYTOX (registered trademark), TO-PRO (registered trademark) -1Iodide, TO-PRO (registered trademark) -3 Iodide, TOTO (registered trademark) -1 Iodide, TOTO (registered trademark) -3 Iodide, YO-PRO (registered trademark) -1 Iodide, YO-PRO (registered trademark) -3 Iodide, YOYO (registered trademark) -1 Iodide, YOYO (registered trademark) -3 Iodide.

In addition, a known dye can be used as a general dye. However, when a dye component having absorption in the wavelength region overlapping with the hemoglobin absorber is included as a staining dye component, there may be a negative effect that the ability to detect hemoglobin decreases, and it may be difficult to identify the desired nucleated red blood cell. is there. Therefore, it is preferable to use a staining dye that does not overlap the absorption wavelength region of hemoglobin as the staining dye component.

In this embodiment, nuclear hematoxylin, toluidine blue, methylene blue, azure blue, cresyl violet, propidium iodide, methyl green, and nuclea fast red can be used. These dyes can be preferably used because they have substantially no adverse effect on the ability to detect hemoglobin.

Staining of cell nuclei can be performed in any step from the preparation step (step S12) to the concentration step (step S16) and after preparation of the specimen.

[Identification of fetal cells]
In the concentration step, fetal cells can be identified by observing a label of an antibody that specifically reacts with fetal cells when concentration is performed using an antigen-antibody reaction.

In the concentration step, when a method using a light source antibody reaction is not used, in the identification step, nucleated red blood cells are identified, and the obtained nucleated red blood cells are genetically analyzed, so that fetal nucleated red blood cells or mothers are analyzed. It can be distinguished from nucleated red blood cells of origin.

[Red blood cell identification]
In the present embodiment, red blood cells and other blood cells can be distinguished using light absorption of hemoglobin present in red blood cells. By irradiating the fraction of maternal blood in which nucleated red blood cells are concentrated in the concentration step with light having a maximum peak wavelength of 380 nm to 470 nm to obtain a transmission image, the red blood cells and cells are hemoglobin. It is possible to distinguish blood cells other than red blood cells that are not absorbed. In addition, morphological observation in a bright field using a blue filter or the like is preferable because stronger confirmation that it is an erythrocyte system can be obtained.

<Acquisition Step (Step S20)>
The nucleated red blood cells identified in the identification step are preferably recovered in the acquisition step, and more preferably only the nucleated red blood cells to be analyzed are isolated and recovered. As a method for isolating and recovering nucleated red blood cells, a known method can be used, and in particular, it is preferable to use a micromanipulation (MM) method or a laser microdissection (LMD) method.

The micromanipulation system can be combined with various micromanipulators of Narishige Co., Ltd., for example. As the laser microdissection system, for example, a commercially available system such as a laser microdissection system PALM MicroBeam manufactured by ZEISS can be used.

<Analysis process (step S22)>
In the analysis step, the amplification step of amplifying the nucleic acid contained in the chromosome of the nucleated erythrocyte isolated and recovered in the acquisition step, the amount of the amplified product of the nucleated erythrocyte is determined, and the nucleated nuclei derived from the fetus by gene analysis It consists of a determination step for confirming the presence of red blood cells and a determination step for determining the presence or absence of a numerical abnormality of fetal chromosomes by comparing the amount of amplified DNA of fetal nucleated red blood cells.

[Amplification process]
The amplification step is a step of amplifying nucleic acid contained in the chromosome of the nucleated red blood cell identified by the identification step or at least the fetal nucleated red blood cell. In the amplification step, DNA is extracted from cells isolated from a microwell plate or a smear, and genome amplification is performed. Genomic amplification can be performed using a commercially available kit.

As the genome amplification method used in this embodiment, the obtained cells are eluted from the cells through a general method of cell lysis using a surfactant, proteolysis step using protease K, etc. The genomic DNA obtained by the above is used.

As a whole genome amplification reagent, reagents based on polymerase chain reaction (PCR: PolymerasePoChain Reaction) PicoPLEX WGA kit (New England Biolabs), GenomePlex Single Cell Whole Genome Amplification kit (Sigma-Aldrich), MALBAC (Multiple Annealing and Looping) -Based (Amplification Cycles) method (published in International Publication WO2012 / 166425A2) can be used. Further, reagents GenomiPhi (GE Healthcare) and REPLI-g (Qiagen) based on strand displacement DNA synthesis reaction can be used in the same manner. In this embodiment, it is preferable to use PicoPLEX WGA kit (New England Biolabs).

The amplification product of DNA obtained by whole genome amplification can be confirmed for amplification by agarose gel electrophoresis or the like. Furthermore, it is preferable to purify the whole genome amplification product using QIAquick® PCR® Purification® Kit (QIAGEN).

In addition, the concentration of the amplification product of DNA obtained by whole genome amplification can be measured using NanoDrop (Thermo Fisher Scientific), Quantus Fluorometer (Promega), BioAnalyzer (Agilent), TapeStation (Agilent). Is possible.

In the amplification step, nucleated red blood cells, or in the case where fetal-derived cells are identified in the identification step, amplify DNA, which is a nucleic acid present at least in the chromosome of fetal-derived nucleated red blood cells. The number of fetal nucleated red blood cells may be at least one, but it is preferable to amplify nucleic acids obtained from a plurality of fetal nucleated red blood cells. In addition, in order to determine the numerical abnormality of the fetal chromosome in the subsequent determination step, the chromosome of the nucleated red blood cell derived from the mother without the numerical abnormality may be selected as a reference for comparing the amount of the amplified product. This is a preferred embodiment. When comparing nucleated erythrocytes derived from the mother, the nucleic acid of the chromosome of the nucleated erythrocytes derived from the mother is also a preferred embodiment.

[Confirmation process]
In the confirmation step, the amount of amplification product of nucleated red blood cells amplified in the amplification step is confirmed, and fetal nucleated red blood cells are confirmed from the nucleated red blood cells by gene analysis, or the fetal origin existence identified in the identification step. This is a step of confirming nuclear red blood cells.

[Gene analysis]
For gene analysis, DNA microarray, digital PCR, next-generation sequencer, and nCounter System (NanoString) can be used. In this embodiment, the accuracy and speed of analysis are the number of samples that can be processed at one time. It is preferable to use a next-generation sequencer from the standpoint of a large number of items.

In the present embodiment, the next-generation sequencer means a sequencer classified in comparison with a capillary sequencer (called a first generation sequencer) using the Sanger method. Next generation sequencers include second generation, third generation, fourth generation, and sequencers that will be developed in the future. The most popular next-generation sequencer at present is a sequencer based on the principle of determining a base sequence by capturing fluorescence or light emission linked to complementary strand synthesis by DNA polymerase or complementary strand binding by DNA ligase. Specific examples include MiSeq (Illumina), HiSeq2000 (Illumina, HiSeq is a registered trademark), Roche454 (Roche).

When analyzing amplification products of DNA obtained in the amplification process using a next-generation sequencer, it is possible to use whole genome sequences, exome sequences, and amplicon sequences.

Burrows-Wheeler ™ Aligner (BWA) can be used as a means for aligning sequence data obtained by a next-generation sequencer, and it is preferable to map sequence data to a known human genome sequence by BWA. Examples of means for analyzing genes include SAMtools and BEDtools, and it is preferable to analyze gene polymorphisms, gene mutations, and chromosome numbers using these analysis means.

《Allele analysis》
After performing whole genome amplification by the amplification step, it is possible to confirm that the nucleated red blood cells are fetal nucleated red blood cells by determining the allele sequence.

A cell identified as a nucleated red blood cell in the identification step or a cell identified as a fetus-derived nucleated red blood cell, amplified by the polymerase chain reaction (amplification step), and subjected to numerical abnormality inspection The amount of amplification product of DNA having a sequence of a predetermined region of 100 to 150 bp (base pair) with respect to the chromosome is determined with a sequencer. In the present embodiment, the chromosomes to be examined are preferably chromosome 13, chromosome 18, chromosome 21, and chromosome X. Fetal nucleated red blood cells usually inherit one pair of chromosomes from their father and mother, and have two chromosomes except for sex chromosomes. By analyzing the alleles of these one set of chromosomes and confirming the presence of the gene derived from the father, it is possible to select whether the nucleated erythrocyte is a fetal nucleated erythrocyte or a maternal nucleated erythrocyte.

Confirmation of the presence of the gene derived from the father is also performed on the mother-derived cell at the same time, and if there is an allele that does not exist in the cell derived from the mother, this allele is recognized as a gene derived from the father. be able to. When a gene derived from a father is confirmed, the nucleated red blood cell can be selected as a fetus-derived nucleated red blood cell. The maternal cell to be subjected to genetic analysis is not particularly limited, but it is preferable to perform DNA analysis from leukocytes present in maternal blood.

Alleles to be analyzed are single nucleotide polymorphisms (SNPs (SNPs): Single Nucleotide Polymorphism) or copy number polymorphisms (CNP (CNPs) Copy Number Polymorphism), tandem repeats (STR: Short Tandem Repeat) It is preferable to do.

Fetal genes inherit a pair of genes from their parents, and genetic information is recorded as a sequence of four types of chemical substances. In the case of humans, there are about 3 billion bases, but there is a sequence portion that varies depending on individuals at a ratio of 1 to 1000-2000, and this is called a single nucleotide polymorphism. If this single nucleotide polymorphism is analyzed and compared with leukocytes, which are maternally derived cells, if the single nucleotide polymorphism sequence can be confirmed in nucleated red blood cells, it will be confirmed that the nucleated red blood cells are of fetal origin. be able to.

The copy number polymorphism and tandem repetitive sequence is a region in which a DNA sequence is one unit and this DNA sequence is repeatedly arranged in series, and this is a repetitive region. Since fetuses inherit copy number variation and tandem repeats from fathers and mothers, nucleated red blood cells that have copy number variation and tandem repeats that differ from maternal white blood cells are fetal nucleated red blood cells. Can be confirmed.

<< Analysis by Y chromosome >>
When the fetus is a boy, it is possible to confirm whether the nucleated red blood cells are nucleated red blood cells derived from the fetus by confirming the presence or absence of the Y chromosome after performing whole genome amplification by the amplification process.

Since the Y chromosome exists only in males, it does not exist in nucleated red blood cells derived from the mother. Therefore, when the fetus is a boy, if the presence of the Y chromosome can be confirmed, the nucleated red blood cells can be confirmed to be fetal nucleated red blood cells.

[Decision process]
The determination step is a step of determining the presence or absence of a numerical abnormality of the fetal chromosome by comparing the amount of the amplified DNA product of fetal nucleated red blood cells determined in the determination step.

As a standard (or reference) for determining the presence or absence of a numerical abnormality of a fetal chromosome, a chromosome other than the target chromosome to be examined for the numerical abnormality is selected and has a predetermined 100-150 bp region sequence The amount of amplification of the DNA amplification product is determined with a sequencer. The reference chromosome (reference chromosome) is an embodiment in which at least one of the chromosomes other than the target chromosome to be examined for the numerical abnormality of the chromosome of fetal nucleated red blood cells, or a maternally derived nucleated red blood cell. Is selected from the embodiments in which the chromosomes present in the identified cells are selected. In the present embodiment, it is preferable to select a chromosome present in a cell identified as a maternally derived nucleated red blood cell.

Next, it is determined whether there is a numerical abnormality in the chromosome derived from the fetus based on the ratio between the amount of amplification product of the DNA of the target chromosome to be examined for the numerical abnormality and the amount of amplification product of the DNA of the reference chromosome. If the fetus is in a normal state, the amount of amplification product of the target chromosome DNA derived from the fetus to be examined for numerical abnormality and the amount of amplification product of the reference chromosome DNA are approximately 1: 1. Expected to be. If there is a numerical abnormality that is a trisomy in which there are three chromosomes if normal, the ratio is expected to be 1.0: 1.5 (or 2: 3).

In addition, prior to the determination step, the amount of the amplification product of the chromosomal DNA derived from the fetus relative to the amount of the chromosomal DNA amplification product derived from the maternal when the normal fetus is pregnant, collected from a plurality of pregnancy mothers in advance. Obtain the distribution of the results of multiple ratios, and the distribution of the results of multiple ratios of the amount of amplified DNA products from the fetus to the amount of amplified DNA from the mother in the mother of the trisomy fetus. It is also possible to set a cut-off value in a region where these two distributions do not overlap and compare the cut-off value with the ratio of the amount of DNA amplification product to determine whether a numerical abnormality exists. Is possible. In this case, if the ratio of the amount of DNA amplification products is less than or equal to the cutoff value, the fetus is normal, and if the ratio is greater than or equal to the cutoff value, the test result is interpreted as a numerical abnormality that is trisomy. Can do.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to this embodiment.

<Example 1>
By the following method, the preparation process (step S12) to the concentration process (step S16) were performed to prepare a smear.

[Preparation process (step S12)]
After giving informed consent from a pregnant pregnant woman, 14 mL of peripheral blood was obtained in two 7 mL vacuum blood collection tubes containing EDTA-2Na (disodium ethylenediaminetetraacetate) as an anticoagulant.

The obtained maternal blood was diluted with 4 ° C. diluent (D-PBS (−) + 0.06 mass% EDTA (ethylenediaminetetraacetic acid) +0.1 mass% BSA (bovine serum albumin; Wako Pure Chemical Industries, Ltd.)). Diluted twice.

The prepared sample was stored at 4 ° C. until the concentration step.

[Fixing Step (Step S14)]
The glutaraldehyde solution was added to the maternal blood diluted 2-fold in the preparation step so that the mass concentration after addition was 0.05%, and roller stirring (10 rpm) was performed at 4 ° C. for 30 minutes.

[Concentration step (step S16)]
A Histopaque (Sigma Aldrich) was used as a density gradient centrifugation medium, and a separation medium having a density of 1.095 g / mL was prepared. The prepared separation medium (3.0 mL) was pipetted into a centrifuge tube.

The upper part of the separation medium injected into the centrifuge tube was overlaid with 5.0 mL of maternal blood that had been fixed for 30 minutes, and centrifuged at 22 ° C. and 1430 rpm for 30 minutes. After completion of the centrifugation, the upper part containing about 2.0 mL of the separation medium was pipetted and transferred to a centrifuge tube to obtain a concentrated maternal blood fraction.

The concentrated maternal blood fraction was dispersed as 15 mL with a diluent, and washed by centrifugation at 22 ° C., 2000 rpm for 5 minutes. After completion of the centrifugation, the supernatant was removed with an aspirator.

The maternal blood fraction from which the supernatant was removed was washed again under the same conditions, and then the supernatant was removed with an aspirator to obtain a 100 μL concentrated fraction.

(Preparation of specimen)
The concentrated liquid fraction was collected with a micropipette, spotted on a slide glass, uniformly smeared by a drag glass method, and air-dried to prepare a maternal blood smear.

The dried smear was immersed in an 80% ethanol aqueous solution saturated with trehalose for 20 minutes and then air-dried. After the smear was dried, it was immersed in a toluidine blue aqueous solution (containing 0.002% by mass toluidine blue and 3% by mass trehalose) for 5 minutes, then immersed in a 3% by mass trehalose aqueous solution for 5 minutes, and then air-dried.

<Example 2>
A sample was prepared in the same manner as in Example 1 except that the glutaraldehyde solution in the fixing step was changed to a paraformaldehyde solution in Example 1.

<Example 3>
By the following method, the preparation process (step S12) to the concentration process (step S16) were performed to prepare a smear.

The obtained maternal blood was diluted at 4 ° C. (D-PBS (−) (Wako Pure Chemical Industries, Ltd.) + 0.06 mass% EDTA + 0.1 mass% BSA (bovine serum albumin; Wako Pure Chemical Industries, Ltd.) ))).

200 μL of anti-CD45 antibody (BD Bioscience) labeled with PE (phycoerythrin) was added to 5.0 mL of maternal blood diluted 2-fold, and incubated at 4 ° C. for 30 minutes.

The prepared sample was stored at 4 ° C. until the concentration step.

[Fixing Step (Step S14)]
After the incubation, the glutaraldehyde solution was added so that the mass concentration after addition was 0.05% by mass, and roller agitation (10 rpm) was performed at 4 ° C. for 30 minutes.

The top of the separation medium injected into the centrifuge tube was overlaid with 5.0 mL of maternal blood that had been fixed for 30 minutes, and centrifuged at 22 ° C., 1430 rpm for 30 minutes. After completion of the centrifugation, the upper part containing about 2.0 mL of the separation medium was pipetted and transferred to a centrifuge tube to obtain a concentrated maternal blood fraction.

For the concentrated liquid fraction, 0.1 mass% Triton X-100 (Sigma Aldrich) was allowed to act at 4 ° C. for 4 minutes.

Subsequently, 200 [mu] L of anti-fetal hemoglobin antibody (Thermo Fisher Scientific) labeled with APC (Allophycocyanin) was added and incubated at 4 [deg.] C. for 30 minutes.

Subsequently, 10 μL of SYTOX (registered trademark) (Thermo Fisher Scientific) was added.

Using the Cell Sorter SH800, the prepared concentrated liquid fraction dispersion was fractionated into a microtube containing 4 μL of D-PBS (−) in advance.

(Preparation of specimen)
The nucleated red blood cell fraction collected in a microtube was collected with a micropipette and spotted on a slide glass, and then evenly smeared by the drag glass method and air-dried to prepare a maternal blood smear.

The dried smear was immersed in an 80% by weight ethanol aqueous solution saturated with trehalose for 20 minutes and then air-dried.

<Example 4>
Concentrated fraction dispersion prepared in Example 3 (liquid after addition of SYTOX 10 μL) was added to a fraction corresponding to nucleated red blood cells using Cell Sorter SH800, and 384 wells containing 4 μL of D-PBS (−) in advance. Sorted into plates.

<Comparative Example 1>
A sample was prepared in the same manner as in Example 1 except that the sample was prepared by changing the order of the fixing step and the concentration step in Example 1.

[Identification Step (Step S18)]
With respect to the samples of Examples 1 to 4 and Comparative Example 1 above, nucleated red blood cell candidate cells were identified. In Examples 1 and 2 and Comparative Example 1, nucleated red blood cells were extracted by confirming the presence of hemoglobin by detecting the nucleus in the bright field and observing the blue filter. Examples 3 and 4 extract fetal hemoglobin by fluorescence observation of APC, nuclei by fluorescence observation of SYTOX (registered trademark), and nucleated red blood cells by confirming the presence of hemoglobin by blue filter observation in bright field did.

Table 1 shows the results of the number of nucleated red blood cells obtained. The number of nucleated red blood cells shown in Table 1 is a comparison of the number of nucleated red blood cells in maternal blood 2.5 mL of a pregnant woman with the numerical value of Comparative Example 1 being 100. Comparing Example 1 and Comparative Example 1, nucleated red blood cells identified in Example 1 in which the fixing step was performed before the concentration step were compared with Comparative Example 1 in which the fixing step was performed after the concentration step. It was confirmed that the number increased. In Example 2 where the cell fixing solution was a paraformaldehyde solution, the number of nucleated red blood cells identified was equivalent to that in Example 1. Furthermore, from the comparison between Example 1 and Example 3, the number of nucleated red blood cells identified by carrying out cell sorting using antigen-antibody reaction after density gradient centrifugation as cell concentration treatment It was confirmed that there will be more.

[Acquisition Step (Step S20)]
The nucleated red blood cells to be isolated were determined from the information obtained by the identification process. Ten cells were collected in a microtube using a micromanipulator from a maternal blood smear (Examples 1 to 3, Comparative Example 1) or a well plate (Example 4).

[Amplification process [whole genome amplification]]
Each isolated cell was subjected to whole genome amplification using a PicoPLEX WGA kit (manufactured by New England Biolabs), and a minute amount of genomic DNA in the cell was amplified about 1 million times according to the description in the manual.

The obtained amplification product was purified using QIAquick PCR Purification Kit (QIAGEN), and then the concentration of the amplification product was measured using Quantus Fluorometer dsDNA System (Promega).

(Multiplex PCR)
Primers used for multiplex PCR were prepared from a plurality of chromosomal positions for the purpose of analyzing fetal cells and analyzing numerical abnormalities of chromosomes.

Primers were prepared so that the PCR-amplified base length of each detection region was 100 to 150 base pairs, and the positions of the primers were designed so that each detection region contained a genetic polymorphism. 46 types of primers were mixed so that the final concentration of each primer was 25 nmol / L.

The multiplex PCR was performed using a Multiplex PCR Assay kit (manufactured by Takara Bio Inc.). As reaction conditions, 10 ng of whole genome amplification product obtained from each of fetal nucleated erythrocyte candidate cells as a template, 8 μL of 46 mixed primers, 0.125 μLμMultiplex PCR Mix1, 12.5 µL Multiplex PCR Mix2 The reaction was carried out with 25 μL final solution with water.

As PCR conditions, after denaturation at 94 ° C. for 60 seconds, the reaction was carried out in 30 cycles of 94 ° C. for 30 seconds, 60 ° C. for 90 seconds, and 72 ° C. for 30 seconds.

The obtained PCR product was purified using QIAquick® PCR® Purification® Kit (manufactured by QIAGEN).

Next, in order to perform a sequence reaction using Miseq, an index sequence for sample identification and P5 and P7 sequences for flow cell binding were added to both ends of the multiplex PCR product.

As primers, D501-F (SEQ ID NO: 47), D701-R (SEQ ID NO: 48), D702-R (SEQ ID NO: 49), D703-R (SEQ ID NO: 50), D704-R (SEQ ID NO: 51), D705 -R (SEQ ID NO: 52) and D706-R (SEQ ID NO: 53) were each used at a concentration of 1.25 μmol / L, and PCR was performed using a Multiplex PCR PCR Assay kit.

As PCR conditions, after denaturation at 94 ° C. for 3 minutes, the reaction was carried out in 5 cycles of 94 ° C. for 45 seconds, 50 ° C. for 60 seconds, 72 ° C. for 30 seconds, 94 ° C. for 45 seconds, and 55 ° C. for 60 seconds. The reaction was performed in 11 cycles of 30 seconds at 72 ° C.

The obtained PCR product was purified using AMPure® XP® Kit (manufactured by BECKMAN® COULTER), and the concentration was measured using BioAnalyzer.

Furthermore, as a more accurate quantification of amplification products, quantification was performed using KAPA Library Quantification Kits manufactured by Nippon Genetics.

(Evaluation of DNA amplification)
In the gene analysis, the presence or absence and size of DNA amplification products obtained by whole genome amplification were confirmed by agarose gel electrophoresis, and six primer bands were expressed for each of 10 cells collected by a micromanipulator. Number was evaluated. Table 1 shows the average value when Comparative Example 1 is 100.

In Example 4 in which cells were handled in a solution in all steps from obtaining maternal blood from a pregnant mother to genetic analysis, gene analysis was improved compared to Examples 1 to 3 and Comparative Example 1. It was confirmed that

Claims

A fixing process for fixing cells of maternal blood collected from the pregnant mother;
A concentration step of concentrating the maternal blood after the fixing step;
An identification step of identifying nucleated red blood cells in the maternal blood concentrated by the concentration step.
The method for obtaining nucleated red blood cells according to claim 1, wherein the cell fixing treatment solution used in the fixing step contains at least one of a glutaraldehyde solution and a paraformaldehyde solution.
The method for obtaining nucleated red blood cells according to claim 1 or 2, wherein the concentration step is performed by density gradient centrifugation.
The method for obtaining nucleated red blood cells according to claim 3, wherein after concentration by the density gradient centrifugation method, a fraction enriched with nucleated red blood cells is obtained by cell sorting using an antigen-antibody reaction.
The identifying step comprises identifying fetal nucleated red blood cells,
The method for obtaining nucleated red blood cells according to any one of claims 1 to 4, which is carried out by identifying an antibody that specifically reacts with fetal cells.
A washing step of washing the maternal blood with a washing solution containing a first cell treatment agent after at least one of the fixing step and the concentration step;
The method for obtaining nucleated red blood cells according to any one of claims 1 to 5, wherein the first cell treatment agent is at least one selected from albumin, polyethylene glycol, polyvinyl alcohol, and polyvinylpyrrolidone.
The washing solution contains a second cell treatment agent,
The method for obtaining nucleated red blood cells according to claim 6, wherein the second cell treatment agent is at least one selected from sucrose, melezitose, trehalose, and arginine.
The method for acquiring nucleated red blood cells according to any one of claims 1 to 7, further comprising an acquisition step of collecting the nucleated red blood cells identified in the identification step.
An analysis step of genetic analysis of nucleated red blood cells obtained by the method for obtaining nucleated red blood cells according to any one of claims 1 to 8,
A method for identifying nucleated red blood cells in which the maternal blood is handled in a solution in all steps from the fixing step to the analysis step.