JP2016166768A - Method of acquiring nucleated erythrocyte candidate cells, and nucleated erythrocyte candidate cell-smear substrate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of acquiring nucleated erythrocyte candidate cells, which allows for reliably acquiring the nucleated erythrocyte candidate cells one by one from a substrate, and a nucleated erythrocyte candidate cell smear substrate.SOLUTION: Disclosed is a method of acquiring nucleated erythrocyte candidate cells including; a concentration step for concentrating nucleated erythrocytes in maternal blood by means of density-gradient centrifugation; a concentration adjustment step for adjusting erythrocyte concentration in a portion of the maternal blood containing nucleated erythrocytes concentrated in the concentration step; a smearing step for smearing a first substrate with the portion adjusted in the concentration adjustment step; a candidate cell identification step for identifying nucleated erythrocyte candidate cells in the portion smeared on the first substrate; and an acquisition step for acquiring the nucleated erythrocyte candidate cells from the first substrate. The erythrocyte concentration in the portion is adjusted to 1.0×10to 8.0×10erythrocytes per 1 mL, inclusive, in the concentration adjustment step. Also disclosed is a nucleated erythrocyte candidate cell smear substrate that is produced by using the above method and features an average center-to-center distance between the erythrocyte cells of 30-160 μm, inclusive.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for obtaining nucleated red blood cell candidate cells, and a nucleated red blood cell candidate cell smear substrate, and in particular, a method for obtaining nucleated red blood cell candidate cells for examining nucleated red blood cells in pregnant maternal blood, and The present invention relates to a nucleated red blood cell candidate cell smear substrate.

  As a prenatal diagnosis, an amniotic fluid test for examining the chromosomes of fetal cells in amniotic fluid by amniocentesis has been performed. However, it has been pointed out that this method has the possibility of miscarriage.

  On the other hand, it has recently been found 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 with blood. Therefore, if maternal blood can be used to reliably analyze fetal cell chromosome DNA (deoxyribonucleic acid) in maternal blood with high reproducibility, a safe prenatal diagnosis without the possibility of miscarriage will be realized. Will be able to.

  However, there are only about one fetal cell (nucleated erythrocyte) present in the maternal blood in one milliliter of maternal blood. Obtaining such a small number of fetal cells (nucleated red blood cells) with certainty is a very big problem in performing prenatal diagnosis using maternal blood. It is also known that maternal nucleated red blood cells are also present in pregnant maternal blood, and it is also safe to separate maternal nucleated red blood cells from fetal nucleated red blood cells. This is a problem to be solved in realizing diagnosis.

  As a method for 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 maternal red blood cell components using density gradient centrifugation. In addition, a technology that separates maternal leukocytes magnetically using an antibody that specifically immunoreacts with proteins on the surface of leukocytes (MACS: Magnetic activated cell sorting), specifically immunoreacts with the gamma chain of fetal hemoglobin Using technology that separates fetal nucleated red blood cells using antibodies and fluorescent dyes (FACS method: Fluorescence activated cell sorting), etc., nucleated red blood cells that are fetal cells in maternal blood are obtained. .

  These methods are useful techniques for increasing the concentration of fetal nucleated red blood cells in the blood and increasing the probability of obtaining target cells. However, since there are still maternal blood cells around fetal nucleated red blood cells, fetal nucleated red blood cells could not be obtained reliably in a short time.

  In addition, a technique for identifying a cell type using optical information and acquiring a desired cell is disclosed. For example, Patent Document 1 below uses a contrast image of cytoplasm and nucleus after staining the cell nucleus and cytoplasm to generate an absorption image of transmitted visible light, irradiating excitation light to form a fluorescence image of the nucleus. Discrimination of nucleated red blood cells is described. Patent Document 2 describes a device for identifying a blood cell type using light having a wavelength of 415 nm near the maximum absorption wavelength of hemoglobin.

  On the other hand, regarding the technique of smearing blood on a substrate, Patent Document 3 describes that the thickness of the smear on the substrate is made uniform and the angle and relative movement speed between the two substrates are adjusted in order to make the cell distribution uniform. How to do is described. Patent Document 4 discloses a method for producing a good smear by measuring properties such as viscosity for each specimen using a blood analyzer and adjusting the amount of the sample dropped, the angle of the pulling glass, and the pulling speed. Is described. Patent Document 5 describes a computer that can freely set smearing conditions according to the characteristics of a specimen.

Special Table 2002-514304 JP 58-115346 A JP 61-45769 A JP-A-3-94159 JP 2006-78239 A

  In prenatal diagnosis, fetal-derived nucleated cells present in maternal blood are identified, DNA is extracted from these cells, and the presence or absence of fetal abnormalities is confirmed from chromosome information. Therefore, it is necessary to select a single fetal cell and perform genetic analysis. Patent Documents 3 to 5 have a description regarding analysis by observation of cells, but the conditions for collecting and analyzing one cell have not been studied. If the smear state of the cells is not stable and cell aggregation is observed, it is not possible to accurately identify the cells on the smear sample. There were cases where it was not possible to obtain.

  The present invention has been made in view of such circumstances, and in performing prenatal diagnosis, in order to reliably extract fetal genes and perform gene analysis, the present invention is optimal for sorting nucleated red blood cells. It is an object of the present invention to provide a method for obtaining nucleated red blood cell candidate cells and a nucleated red blood cell candidate cell smearing substrate capable of preparing a specimen and reliably obtaining nucleated red blood cell candidates one by one from the substrate.

In order to achieve the above object, the present invention adjusts the concentration of nucleated red blood cells in maternal blood by density gradient centrifugation and the concentration of blood cells in the maternal blood fraction in which the nucleated red blood cells are concentrated by the concentration step. A concentration adjusting step, a smearing step for smearing the fraction adjusted in the concentration adjusting step on the first substrate, and a candidate cell for identifying candidate cells for nucleated red blood cells from the fraction smeared on the first substrate An identification step and an acquisition step of acquiring nucleated red blood cell candidate cells from the first substrate, and the concentration adjustment step has a blood cell concentration of 1.0 × 10 ⁷ or more per 8.0 mL of fraction 8.0. Provided is a method for obtaining nucleated red blood cell candidate cells adjusted to 10 ⁷ or less.

According to the method for obtaining nucleated red blood cell candidate cells of the present invention, the blood cell concentration of the maternal blood fraction concentrated in the concentration step is adjusted to 1.0 × 10 ⁷ or more and 8.0 × 10 ⁷ or less. Thus, the blood cell concentration smeared on the first substrate can be made uniform. By making the blood cell concentration of the fraction smeared on the first substrate uniform within the above range, aggregation of blood cells on the first substrate can be prevented, so that the target cells are identified. The target cell can be reliably acquired in the acquisition step. In the present invention, the “fraction” refers to a liquid layer divided into a plurality of layers by density gradient centrifugation, particularly a layer containing nucleated red blood cells.

  In another aspect of the present invention, in the smearing step, the amount of smearing the fraction on the first substrate is preferably 1.0 μL or more and 4.0 μL or less with respect to one first substrate.

  According to this aspect, the amount of the fraction to be smeared on the first substrate is 1.0 μL or more and 4.0 μL or less with respect to one first substrate, so that it is uniform on the first substrate. In addition, blood cell components can be smeared.

  In another aspect of the present invention, the smearing step includes a step of dropping a fraction on the first substrate, a second substrate, a first substrate and a first substrate on the fraction on the first substrate. A state in which the angle between the second substrate and the second substrate is 10 ° or more and 60 ° or less and the contact is made with a force of 0.5N or more and 2.5N or less, and the second substrate is in contact with the first substrate And the step of spreading the fraction to the contact position between the first substrate and the second substrate, and the contact between the first substrate and the second substrate at a speed of 0.5 mm / sec to 300 mm / sec. And a step of smearing a fraction on the first substrate by moving the second substrate on the first substrate with the angle and force at the time.

  According to this aspect, the force, angle, and speed at which the second substrate is moved on the first substrate in the smearing process are within the above range. By doing so, blood cell components can be uniformly smeared on the first substrate, and aggregation of blood cells can be prevented. In addition, since smearing can be performed without breaking the shape of the cells, the accuracy of blood cell analysis can be improved. Therefore, each cell can be reliably identified, and the target cell can be reliably obtained.

In another aspect of the present invention, the concentration adjustment step preferably adjusts the blood cell concentration to 2.0 × 10 ⁷ or more and 5.0 × 10 ⁷ or less per 1 mL fraction.

  This aspect further defines a preferable concentration range in the concentration adjusting step. By setting the above range, aggregation of blood cells is suppressed, and the number of blood cells existing on the first substrate is also set to a desired number. be able to. Therefore, it is possible to efficiently search for nucleated red blood cells, and to reliably identify and acquire cells one by one.

  In another aspect of the present invention, the candidate cell identification step is preferably identified based on at least one of cell shape and optical properties.

  In this embodiment, the identification method of the candidate cell identification step is limited. By performing identification based on at least one of the cell shape and the optical property, the nucleated red blood cell can be converted into the optical property by the cell shape. Thus, red blood cells and white blood cells or nucleated red blood cells can be distinguished from maternal or fetal origin.

In order to achieve the above object, the present invention provides a concentration step of concentrating nucleated red blood cells in maternal blood by density gradient centrifugation, and a blood cell concentration of a fraction of maternal blood in which nucleated red blood cells are concentrated by the concentration step, A concentration adjusting step for adjusting the concentration to 1.0 × 10 ⁷ or more and 8.0 × 10 ⁷ or less per mL of the fraction; a smearing step for smearing the fraction adjusted in the concentration adjusting step on the first substrate; A candidate cell identifying step for identifying candidate cells for nucleated red blood cells from a fraction smeared on a first substrate, and a nucleated red blood cell candidate cell smeared substrate for smearing nucleated red blood cell candidate cells, A nucleated red blood cell candidate cell smear substrate in which the average distance between the centers of blood cells contained in the fraction smeared on the substrate is 30 μm or more and 160 μm or less.

According to the nucleated erythrocyte candidate cell smear substrate of the present invention, the concentration of the fraction containing nucleated erythrocytes concentrated in the concentration step is adjusted to 1.0 × 10 ⁷ or more and 8.0 × 10 ⁷ or less, and smear is performed. By producing the substrate, the average distance between the centers of the blood cells contained in the fraction smeared on the first substrate can be set to a distance of 30 μm or more and 160 μm or less. Therefore, it is possible to reliably identify one cell at a time and reliably obtain nucleated red blood cell candidate cells. The “average distance between the centers of blood cells” refers to the distance between the centers of the cells smeared on the smear substrate by circular approximation or ellipse approximation.

  In another aspect of the present invention, in the smearing step, the amount of smearing the fraction on the first substrate is preferably 1.0 μL or more and 4.0 μL or less for one first substrate.

  According to this aspect, the amount of the fraction to be smeared on the first substrate is 1.0 μL or more and 4.0 μL or less with respect to one first substrate, so that it is uniform on the first substrate. In addition, blood cell components can be smeared.

  In another aspect of the present invention, the smearing step includes a step of dropping a fraction on the first substrate, a second substrate, a first substrate and a first substrate on the fraction on the first substrate. A state in which the angle between the second substrate and the second substrate is 10 ° or more and 60 ° or less, and the force is 0.5N or more and 2.5N or less, and the second substrate is in contact with the first substrate And the step of spreading the fraction to the contact position between the first substrate and the second substrate, and the contact between the first substrate and the second substrate at a speed of 0.5 mm / sec to 300 mm / sec. And a step of smearing a fraction on the first substrate by moving the second substrate on the first substrate with the angle and force at the time.

  According to this aspect, the force, angle, and speed at which the second substrate is moved on the first substrate in the smearing process are within the above range. By doing so, blood cell components can be uniformly smeared on the first substrate, and aggregation of blood cells can be prevented. In addition, since smearing can be performed without breaking the shape of the cells, the accuracy of blood cell analysis can be improved. Therefore, each cell can be reliably identified, and the target cell can be reliably obtained.

  According to the method for obtaining nucleated red blood cell candidate cells of the present invention and the nucleated red blood cell candidate cell smear substrate, by prescribing the blood cell concentration of the fraction containing the nucleated red blood cell candidate cells before smearing on the substrate, An optimal specimen for identifying nucleated erythrocytes can be prepared, and nucleated erythrocyte candidate cells can be reliably analyzed and acquired one by one.

It is the flowchart figure which showed the procedure of the acquisition method of a nucleated erythrocyte candidate cell. It is a figure explaining a smearing process. It is the flowchart figure which showed the procedure which sort | selects with the shape of a cell (shape of a nucleus). It is the schematic diagram (a) which shows the shape of the cell to select, and the schematic diagram (b) which shows the shape of the cell to remove. And reduced hemoglobin (Hb), which is a graph showing an absorption coefficient for the wavelength of oxyhemoglobin (HbO _2).

  Hereinafter, a method for obtaining nucleated red blood cell candidate cells and a nucleated red blood cell candidate cell smear substrate according to 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 obtaining nucleated red blood cell candidate cells. The method for obtaining fetal nucleated red blood cell candidate cells includes a collection step (step S12) for collecting maternal blood from a pregnant mother, and a concentration step (step S14) for concentrating nucleated red blood cells in the maternal blood by density gradient centrifugation. ), A concentration adjustment step (step S16) for adjusting the blood cell concentration of the fraction of the maternal blood in which the nucleated red blood cells are concentrated by the concentration step, and a fraction of the maternal blood containing the nucleated red blood cells whose concentration has been adjusted A candidate for identifying nucleated red blood cell candidate cells from the smearing step (step S18) for smearing on one substrate and image information (cell shape) and optical information (optical properties) from the fraction smeared on the substrate A cell identification step (step S20), and an acquisition step (step S22) for selectively acquiring nucleated red blood cell candidate cells identified by the candidate cell identification step from the first substrate. The method for acquiring nucleated red blood cell candidate cells of the present invention is a process from the concentration step (step S14) to the acquisition step (step S22).

<Collecting process (step S12)>
The collecting step is a step of collecting maternal blood that is a blood sample. The maternal blood is preferably peripheral blood of a pregnant mother who is not likely to invade.

  Maternal peripheral blood includes maternal eosinophils, neutrophils, basophils, mononuclear cells, lymphocytes and other white blood cells, and mature erythrocytes without nuclei, as well as maternal nucleated red blood cells, And fetal nucleated red blood cells are included. 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.

Fetal nucleated red blood cells are red blood cell precursors that pass through the placenta and are present in the maternal blood. When the mother is pregnant, fetal red blood cells can be nucleated. 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 1 in 10 ⁶ cells in maternal blood, and the existence probability is very low in the maternal peripheral blood.

<Concentration step (step S14)>
Next, the nucleated red blood cells in the maternal blood collected in the collection step are concentrated by a concentration step by density gradient centrifugation.

[Density gradient centrifugation]
The density gradient centrifugation method is a method of separation based on the difference in density of components in blood, and is selected by selecting the first medium and the second medium so as to sandwich the density values of the components to be separated. The target component (in this embodiment, fetal nucleated red blood cells) can be collected at the interface between the first medium and the second medium later. And the blood which concentrated the nucleated red blood cell can be obtained by extract | collecting the fraction which exists in the interface between this 1st medium and the 2nd medium.

  International Publication No. WO2012 / 023298 describes the density of maternal blood including fetal nucleated red blood cells. According to the description, the expected density of fetal nucleated red blood cells 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. Acid spheres 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, lymphocytes are 1 About 0.060 to 1.080 g / mL, and mononuclear cells are about 1.060 to 1.070 g / mL.

  The density of the medium to be laminated (first medium and second medium) is to separate fetal nucleated red blood cells having a density of about 1.065 to 1.095 g / mL from other blood cell components in the mother's body. To be set. Since the density of the center of nucleated red blood cells derived from a fetus is about 1.080 g / mL, two different density media sandwiching this density are created, and adjacent layers are layered to form a desired fetus at the interface. It is possible to collect nucleated red blood cells derived from them. Preferably, the density of the first medium is set to 1.08 g / mL or more and 1.10 g / mL or less, and the density of the second medium is set to 1.06 g / mL or more and 1.08 g / mL or less. More preferably, the density of the first medium is 1.08 g / mL or more and 1.09 g / mL or less, and the density of the second medium is 1.065 g / mL or more and 1.08 g / mL or less. As a specific example, by setting the density of the first medium to 1.085 g / mL and the density of the second medium to 1.075 g / mL, plasma components, eosinophils, and mononuclear cells are added. It is possible to separate the desired fraction to be collected. It is also possible to separate some of red blood cells, neutrophils, and lymphocytes. In the present invention, the first medium and the second medium may be the same type or different types, as long as the effects of the present invention can be realized. However, it is preferable to use the same type of medium.

  In the present embodiment, the first medium and the second medium having different densities for density gradient centrifugation are Percoll (registered trademark), which is a colloidal particle dispersion of 15-30 nm in diameter and coated with polyvinylpyrrolidone. Use media such as Ficoll®-Paque, a neutral hydrophilic polymer rich in side chains made from sucrose, Histopaque®, a solution of polysucrose and sodium diatrizoate Can do. In the present embodiment, it is preferable to use Percoll (registered trademark) and Histopaque (registered trademark). For Percoll (registered trademark), a product having a density (specific gravity) of 1.130 is commercially available, and a medium having a target density (specific gravity) can be prepared by dilution. Histopaque (registered trademark) can prepare the first medium and the second medium to a desired density using a commercially available medium having a density of 1.077 and a medium having a density of 1.119. .

<Density Adjustment Step (Step S16)>
The concentration adjustment step is a step of adjusting the blood cell concentration of the maternal blood fraction in which the nucleated red blood cells obtained in the concentration step are concentrated.

  Nucleated erythrocyte candidate cells that collect at the interface of media with different densities can be obtained by the concentration step by density gradient centrifugation. The desired fraction of maternal blood obtained by the concentration process is mainly composed of blood cells, but there are differences in viscosity due to individual differences in the mother, disturbances in the interface of the density medium for sampling, differences in the temperature of the working environment, etc. Due to the difference in the viscosity of the medium, it is very difficult to control the blood cell concentration of the obtained fraction uniformly for each operation. Therefore, if the number of cells on the substrate changes when they are smeared on the substrate as they are, the cells may overlap or the interval between cells may become very large, which may hinder subsequent analysis and cell acquisition. Will increase. Therefore, in order to uniformly smear blood cells even under conditions such as room temperature and different patients, the blood cell concentration of the maternal blood fraction solution containing blood cells sampled after the concentration step is adjusted. By adjusting the blood cell concentration of the maternal blood fraction solution, the density of the cells smeared on the substrate is kept constant, preventing aggregation of blood cells on the substrate to be produced, and uniform Substrate fabrication and reliable analysis can be realized. Moreover, by performing the analysis reliably, cells that are fetal nucleated red blood cell candidates can be identified, and cells that are fetal nucleated red blood cell candidates can be reliably obtained one by one from the substrate. it can.

  To adjust the blood cell concentration, first, the blood cell concentration of a sample obtained by separating red blood cells and nucleated red blood cells in the concentration step is measured with a cell counter, and the dilution rate is calculated so that the blood cell concentration falls within the target range. Based on this calculated dilution factor, the blood cell concentration is adjusted using the diluent.

As a range of blood cell concentration to be adjusted by the concentration adjusting step, the blood cell concentration is preferably adjusted to 1.0 × 10 ⁷ or more and 8.0 × 10 ⁷ or less per mL, and preferably 1.5 × 10 5 per mL. it is more preferable to adjust the ^seven or more 5.5 × 10 ⁷ or less, and more preferably adjusted to 2.0 × 10 ⁷ or more per 1 mL 5.0 × 10 ⁷ or less. By adjusting the blood cell concentration within this range, it is possible to realize a substrate in which the average distance of cells on the substrate is 30 μm or more and 160 μm or less, and there are few cells to aggregate. Therefore, it becomes possible to acquire nucleated red blood cell candidate cells one by one from the substrate.

  As a method for measuring the blood cell concentration, for example, it can be measured by using a fully automatic cell counter TC20 manufactured by Bio-Rad. After the maternal blood is collected, 10 μL of a blood sample containing concentrated nucleated red blood cell candidates is obtained with a pipette and sealed in a measurement substrate dedicated to the fully automatic cell counter TC20. When this dedicated substrate is inserted into the TC 20, the blood cell concentration is automatically measured. Based on this value, the dilution factor is calculated so that the blood cell concentration falls within the target range, and the blood cell concentration is adjusted using the diluent based on the calculated dilution factor.

  As a diluent used for adjusting the blood cell concentration of a solution containing blood cells to be smeared on the substrate, it is preferable to use a phosphate buffered saline (PBS).

<Smearing step (step S18)>
Next, the fraction of maternal blood whose concentration has been adjusted in the concentration adjusting step is smeared on the first substrate. The smearing step can be performed by a drawing glass method (wedge method), a crushing method (crushing method), a hand stretching method, a spin method, etc., and it is particularly preferably performed by a drawing glass method. FIG. 2 is a diagram illustrating a smearing process in which smearing is performed by a pulling glass method. The smearing step is a step of dropping the fraction 16 of the maternal blood whose concentration has been adjusted onto the first substrate 12 using the dropping means 14 (FIG. 2A), and the fraction 16 on the first substrate 12. A step of bringing the second substrate 18 into contact with the first substrate 12 (FIG. 2B), a step of spreading the fraction 16 dropped onto the contact position between the first substrate 12 and the second substrate 18 (FIG. 2C), There is a step (FIG. 2 (d)) of smearing the fraction 16 on the first substrate 12 by moving the second substrate 18 on the first substrate 12 at a predetermined speed.

[Step of dropping (FIG. 2 (a))]
First, the fraction 16 whose concentration has been adjusted in the concentration adjusting step is dropped onto the end of the first substrate 12 using the dropping means 14. As the first substrate 12, a transparent substrate is preferable, and specifically, a slide glass is preferable. The amount dropped is preferably 1.0 μL or more and 4.0 μL or less, and more preferably 1.5 μL or more and 2.5 μL or less with respect to one first substrate. If the amount of the fraction (blood) used as the smear is 1.0 μL or more, the amount is appropriately present. Therefore, the second substrate 18 is brought into contact with the fraction 16 in the subsequent step, and the fraction 16 When the layer is spread by capillarity, the fraction 16 is sufficiently widened and smearing can be performed uniformly. If it is less than 1.0 μL, the amount is small, so that the smear region is limited to a part of the first substrate 12 and the first substrate 12 cannot be used efficiently, and the smear to be inspected. There are cases where the number of substrates increases and it takes a very long time for inspection. In addition, when the amount is 4.0 μL or less, the amount of the liquid in the fraction can be set to an appropriate amount, and at the end of the smearing process, there is no liquid left on the first substrate 12 and the smeared area is removed. A smear substrate having a uniform blood cell component is formed in the latter half (the direction in which the second substrate is moved). Even when the target nucleated red blood cell is present in the latter half of the smear region, it becomes possible to easily identify and acquire cells.

[Step of contacting (FIG. 2B)]
Next, the second substrate 18 is brought into contact with the fraction 16 on the first substrate 12. As shown in FIG. 2B, the position where the second substrate 18 is brought into contact is brought into contact with the end portion in the direction in which the fraction 16 dropped on the first substrate 12 is expanded. The second substrate 18 is preferably a transparent substrate, specifically a slide glass, like the first substrate 16.

  The second substrate 18 is preferably in contact with the tip of the second substrate 18 at an angle θ of 10 ° or more and 60 ° or less with respect to the first substrate 16. More preferably, it is 25 ° or more and 40 ° or less. Further, the force for bringing the second substrate 18 into contact with the first substrate 16 is preferably 0.5 N or more and 2.5 N or less, more preferably 1.5 N or more and 2.0 N or less. By setting the angle and force when the first substrate 12 and the second substrate 18 are in contact with each other within the above range, the blood cell component can be smeared uniformly. Moreover, since the shape of a cell can also be maintained, the accuracy of cell image analysis can be improved, and cells can be obtained.

[Step of spreading (FIG. 2 (c))]
After the second substrate 18 is brought into contact with the first substrate 12, the second substrate 18 is held in that state, and the first substrate 12 is brought into contact with the first substrate 12 at the contact position where the second substrate 18 and the first substrate 12 are in contact. The maternal blood fraction 16 is spread in a space surrounded by the substrate 12 and the second substrate 18. It is preferable that the maternal blood fraction 16 spreads along the pressed contact position (direction passing through the paper surface in FIG. 2) and utilizes the capillary phenomenon as the spreading force. In order to sufficiently spread the dropped fraction 16 between the first substrate 12 and the second substrate 18, the second substrate 18 is kept in contact with the first substrate 12 for 2 to 5 seconds. It is preferable.

[Step of smearing (FIG. 2 (d))]
After the fraction 16 is spread between the second substrate 18 and the first substrate 16, the second substrate 18 is moved on the first substrate 12 at a constant speed, whereby the first substrate 12 is moved. Spread fraction 16 on top and smear blood cells. By moving the fraction 16 sufficiently wet and spread between the second substrate 18 and the first substrate 12 at a constant speed, the fraction 16 of the maternal blood whose blood cell concentration is adjusted on the first substrate 12 is obtained. Can be smeared uniformly. The moving speed of the second substrate 18 is preferably moved at a constant speed, and the value of the speed is preferably 0.5 mm / sec or more and 300 mm / sec or less, more preferably 50 mm / sec or more and 250 mm / sec or less. More preferably, it is 100 mm / sec or more and 200 mm / sec or less.

  On the substrate thus prepared (FIG. 2 (e)), the average distance between the centers of blood cells contained in the maternal blood fraction 16 can be 30 μm or more and 160 μm or less. Since the average size of blood cells is about several tens of μm, in order to obtain one cell from the first substrate 12, it is necessary to avoid the cells from overlapping each other. It is preferable to have 30 μm or more. Furthermore, the average distance between the centers of blood cells is preferably 40 μm or more and 120 μm or less. By using a smear substrate in which the average distance between the centers of blood cells is in the above range, it becomes possible to perform analysis on each cell with certainty. Candidate cells for nucleated red blood cells can be obtained.

<Candidate cell identification step (step S20)>
Next, analysis is performed on maternal blood cells smeared on the first substrate 16 to identify nucleated red blood cell candidate cells. By confirming that the nucleated red blood cell candidate cells are present on the first substrate by the candidate cell identification step, a nucleated red blood cell candidate cell smeared substrate on which the nucleated red blood cell candidate cells are smeared can be produced.

  In the present invention, as a preferable analysis method, from the form of cells, the form of cell nuclei, from the method of analyzing nucleated red blood cells, or from the information of hemoglobin present in the cytoplasm of red blood cells having a large absorption in the wavelength range of 380 nm to 420 nm, Perform analysis using methods such as separation of leukocytes and red blood cells, separation methods using optical information of fetal globin, and labeling and separation of anti-fetal hemoglobin antibodies that specifically interact with fetal globin. Is preferred.

  Hereinafter, as one aspect of the analysis method, a method for identifying nucleated red blood cell candidates by image analysis will be described. In this embodiment, nucleated erythrocyte candidate cells are selected by selecting according to information based on cell shape and then selecting according to optical characteristics.

(Selection based on cell shape information)
Examples of the selection based on the cell shape include the ratio of the area of the nucleus region to the area of the cytoplasm, the circularity of the nucleus, the area of the nucleus region, the brightness of the nucleus, and how the nucleus collapses. In particular, it is preferable to select cells having conditions satisfying the ratio of the area of the nuclear region to the area of the cytoplasm and the circularity of the nucleus as fetal nucleated red blood cell candidates. The area of the cytoplasm and the area of the nucleus region are the areas on the observed surface of the specimen for analysis.

  FIG. 3 is a flowchart showing an example of a procedure for sorting by cell shape (nucleus shape) in the nucleated red blood cell sorting step.

  In the selection based on the shape of the cell, first, the cell is extracted (step S32). Judgment whether it is a cell is performed using a binarized image. A brightness information of an image obtained by photographing a sample for analysis using a white light source is used to generate a binarized image using a predetermined threshold value, and dot-like and island-like shapes are generated from the generated binarized image. A cell having a continuous region, that is, a region closed by an arbitrary closed curve is extracted as a cell. Note that the binarized image may be subjected to morphological processing such as Erosion / Dilation to adjust the shape of the continuous region and then the size may be obtained.

  Next, the cells extracted in step S32 are selected based on the presence or absence of nuclei (step S34). The presence or absence of nuclei is selected depending on whether nuclei (dots) are present in the cells. A binarized image can also be used to determine the presence or absence of a nucleus. When there is a region closed by an arbitrary closed curve having an area smaller than the region extracted as a cell in the region extracted as a cell, the region can be extracted as a nucleus.

  Next, cell nuclei with a size of 1 to 5 μm are extracted (the size of the dotted and island-like continuous regions) (step S36). Selecting cells having nuclei with a size of 1-5 μm is effective for sorting nucleated red blood cells. Note that “the size of the dotted and island-like continuous regions” means that the closed curve representing the outer shape of the nucleus is approximated to a circle or an ellipse, and the circular diameter approximating the outer shape of the nucleus or the outer shape of the cell is approximated. The major axis of the ellipse can be made.

  As an example of a circular shape that approximates the outer shape of the nucleus, a circular shape that circumscribes the closed curve that represents the outer shape of the nucleus has the smallest diameter. As an example of an ellipse that approximates the outer shape of the nucleus, an ellipse that circumscribes the closed curve representing the outer shape of the nucleus and that has the smallest major axis can be cited. A circular diameter approximating the outer shape of the nucleus or an ellipse having a major axis of 1 to 5 μm approximating the outer shape of the nucleus is extracted.

Next, the cells are sorted according to the shape of the nucleus (step S38). FIG. 4A is a schematic diagram showing the shape of a cell to be selected, and FIG. 4B is a schematic diagram showing the shape of a cell to be excluded. As specific examples of the selection based on the shape of the nucleus, a method for performing selection based on the ratio of the area of the nucleus region to the area of the cytoplasm and a method for performing selection based on the circularity of the nucleus will be described. When the ratio of the area of the nuclear region to the area of the cytoplasm satisfies the following formula (1), it is selected as a nucleated red blood cell that is a candidate for a fetal nucleated red blood cell. When the cell cytoplasm area is C and the cell nucleus area is N, cells having nuclei with a ratio of N to C exceeding 0.25 and less than 1.0 are fetal nucleated red blood cell candidates. Selected as nucleated red blood cells.
0.25 <(N / C) <1.0 (1)

Moreover, as a case where the selection condition for the degree of circularity of the nucleus is satisfied, a case where the following expression (2) is satisfied can be cited. When the nucleus diameter or the major axis of the nucleus is L, the ratio of the area of the nucleus to the square of the nucleus diameter or the major axis of the nucleus is greater than 0.65 and less than 0.785. Selected as a nucleated red blood cell candidate for red blood cell. Note that the diameter of the nucleus or the major axis of the nucleus can be obtained by the same method as that for obtaining the nucleus size in step S44.
0.65 <(N / (L × L)) <0.785 (2)

  Examples of the system configuration for selecting nucleated red blood cells that are candidates for fetal nucleated red blood cells using cell shape information include an optical microscope, a digital camera, a slide glass stage, an optical transport system, an image processing PC ( personal computer), a control PC, and a system equipped with a display. The optical transport system includes an objective lens and a CCD (Charge Coupled Device) camera, and the image processing PC includes a processing system that performs data analysis and data storage. The control PC includes a configuration including a position control of the slide glass stage and a control system for controlling the entire processing.

(Sorting by optical characteristics)
The sorting based on the optical characteristics can utilize spectral characteristics resulting from the difference in oxygen affinity of hemoglobin contained in red blood cells. FIG. 5 is a graph showing absorption coefficients with respect to wavelengths of reduced hemoglobin (Hb) and oxidized hemoglobin (HbO ₂ ) (described in JP-A-2014-1485). Here, the absorption coefficient is a constant indicating how much light the medium absorbs when the light enters the medium. As shown in FIG. 5, it is known that the absorption coefficient of hemoglobin changes due to the interaction with oxygen. That is, oxyhemoglobin has a red color tone, and becomes bluish as it becomes reduced hemoglobin.

  In sorting by optical properties, fetal nucleated red blood cells are derived from maternal erythrocytes by utilizing the difference in oxygen affinity of hemoglobin with respect to maternal erythrocytes and slightly existing maternal nucleated red blood cells. Nucleated red blood cells and fetal nucleated red blood cells can be selected. For example, by using a blood component coated on a glass substrate, the difference in spectral characteristics between nucleated red blood cells and red blood cells present in the vicinity of the nucleated red blood cells can be used to obtain maternal nucleated red blood cells and fetuses. The nucleated red blood cells are selected.

  The spectral characteristic is to detect a difference in a wavelength region where there is a difference in absorption coefficient between reduced hemoglobin and oxidized hemoglobin. Hemoglobin in adult erythrocytes is tetramer α2β2 (HbA). On the other hand, hemoglobin in fetal erythrocytes is a tetramer α2γ2 (HbF) made up of two α chains and two γ chains, and is converted into HbA2 consisting of HbA, which occupies most and a small number of HbF after birth. It is known to replace it. In the placenta, the fetal hemoglobin obtains oxygen by receiving oxygen from the maternal hemoglobin (HbA) in the blood. And hemoglobin (HbA) contained in red blood cells in adult blood is tetramer α2β2, but fetal hemoglobin (HbF) is α2γ2, and has a characteristic of higher oxygen affinity than HbA. . In the prenatal diagnosis, it is preferable to perform a test by collecting peripheral blood of the pregnant mother as a blood sample. Peripheral blood is usually collected from veins, and the amount of oxygen in the veins is less than the amount of oxygen in the arteries. In healthy people, the oxygen saturation of central venous blood is said to be 60-80% normal, and this value fluctuates depending on the oxygen demand throughout the body. Since HbA and HbF have different oxygen affinity, the amount of oxygen binding between HbA and HbF is different even in venous blood, and the amount of oxygen binding between HbF is higher than that of HbA. .

  Maternal hemoglobin (HbA) and fetal hemoglobin (HbF) also have a difference in oxygen affinity in the vein. As shown in FIG. 5, there is a difference in absorption coefficient between reduced hemoglobin and oxidized hemoglobin. Sorting is performed using the difference in absorbance between nucleated red blood cells derived from fetuses and nucleated red blood cells derived from fetuses. A light wavelength region of 400 to 650 nm is applied to the measurement of absorbance. The absorbance is measured using at least one monochromatic light having a wavelength selected from this light wavelength range.

Absorbance can be measured more reliably by measuring the absorbance of each wavelength using multiple monochromatic lights of different wavelengths and detecting the ratio of the measured values of each wavelength. . Preferably, it is an embodiment using monochromatic light having a wavelength selected from a light wavelength range of 400 to 500 nm, more preferably monochromatic light having a wavelength selected from a light wavelength range of more than 450 nm and less than 480 nm, and more than 550 nm It is preferable to use a monochromatic light having a wavelength selected from a light wavelength region of less than 575 nm, respectively, and to sort by the ratio of absorbance at each wavelength. In addition, the light wavelength range is a monochromatic light having a wavelength selected from a light wavelength range exceeding 550 nm and less than 575 nm, and a monochromatic light having a wavelength selected from an optical wavelength range exceeding 575 nm and less than 585 nm. It is preferable to select according to the ratio of the absorbance. By deriving the ratio of absorbance measured by two or more different monochromatic lights, it is possible to eliminate absorbance errors caused by cells such as cell thickness and area between individual cells. As shown in FIG. 5, these light wavelength regions have a large difference in absorption coefficient between oxygenated hemoglobin (HbO ₂ ) and reduced hemoglobin (Hb). Sorting between nuclear red blood cells and fetal nucleated red blood cells can be performed reliably.

  In addition, the optical wavelength range selected according to the optical characteristics is the wavelength of each optical wavelength region with the absorption coefficient of the graph shown in FIG. 5 sandwiching the wavelength where the absorption coefficient of reduced hemoglobin and the absorption coefficient of oxyhemoglobin are reversed. It is preferable to measure. That is, the wavelength of the first light wavelength region in which the absorption coefficient of fetal nucleated red blood cells (the ratio of oxygenated hemoglobin is high) is higher than the absorption coefficient of maternally derived nucleated red blood cells (the ratio of oxygenated hemoglobin is low); The absorbance is measured at a wavelength in the second light wavelength region where the absorption coefficient of the nucleated red blood cells derived from the mother is higher than the absorption coefficient of the nucleated red blood cells derived from the fetus. And by calculating | requiring and comparing the ratio of the light absorbency measured in the wavelength of the 1st light wavelength range, and the light absorbency measured in the wavelength of the 2nd light wavelength range, the ratio of the nucleated erythrocyte derived from a fetus and the maternal origin Since the difference in the proportion of nucleated red blood cells can be increased, fetal nucleated red blood cells can be reliably selected.

  Examples of the first light wavelength region include a light wavelength region exceeding 400 nm and less than 500 nm, exceeding 525 nm and less than 550 nm, and exceeding 575 nm and less than 585 nm. In addition, examples of the second light wavelength region include a light wavelength region of more than 550 nm and less than 575 nm.

  It is also possible to measure the absorbance at a plurality of wavelengths in each light wavelength region and perform selection using the average value of the absorbances. By selecting based on the average value of absorbance, the influence of noise included in the measured value of absorbance can be reduced.

  As a method of selecting nucleated red blood cells derived from fetuses and nucleated red blood cells derived from the mother by spectral characteristics, red blood cells that do not have nuclei around nucleated red blood cells to be selected are used as reference red blood cells. By comparing, it is selected whether the nucleated red blood cells to be selected are fetal-derived nucleated red blood cells or maternally-derived nucleated red blood cells. By measuring the absorbance of the reference erythrocytes with monochromatic light having the same wavelength as the nucleated red blood cells to be sorted, determining the ratio, and comparing the absorbance ratio of the nucleated red blood cells to be sorted with the absorbance ratio of the reference red blood cells, Fetal nucleated red blood cells can be more reliably selected from nucleated red blood cells to be sorted.

<Acquisition process (step S22)>
Returning to FIG. 1, in the acquisition step, the nucleated red blood cell candidate cells derived from the fetus and the nucleated red blood cell candidate cells identified as mother-derived nucleated red blood cells in the candidate cell identification step are selectively selected from the first substrate. It is the process of acquiring one by one. For obtaining cells, a laser microdissection device or a micromanipulator can be used.

(Laser microdissection)
The laser microdissection is an apparatus system that can cut and process a minute region by combining a laser such as a commercially available Zeiss PALM Micro Beam with a laser. Laser dissection is a device that can selectively cut out only the target part of a sample smeared on a substrate (slide glass or glass slide with a polymer coat on it). is there. For this reason, it is preferable that cells or tissues are uniformly applied to the substrate. Thereby, it becomes possible to acquire only the target single cell.

  In the acquisition step of acquiring nucleated red blood cell candidate cells from the substrate, the positional information of the cells on the substrate sorted according to the spectral characteristics is acquired in advance, and the positional information is input to the laser microdissection device. When the substrate is set in the laser microdissection device, the device automatically moves to the target position based on the position information, acquires only the target cells with the laser according to the laser output conditions registered in advance, Sort cells into collection tube.

(Micromanipulator)
A micromanipulator is a device capable of handling cells under a microscope. For example, a Narishige microinjection system or a micromanipulator system is used in a form that can be added to a commercially available microscope system. Any single cell can be obtained from a sample substrate smeared with a target cell under a microscope.

  In the acquisition process of acquiring nucleated red blood cell candidate cells from the substrate, the positional information of the cells on the substrate sorted according to the spectral characteristics is acquired in advance as in the laser microdissection, and the position coordinates are input to the micromanipulator system. To do. The substrate set in the micromanipulator system moves to the target cell acquisition position based on the position information. Thereafter, the manipulator is operated to obtain only the target cells and sealed in a tube containing the cell recovery solution. When analyzing the DNA of cells enclosed in a tube, the cell recovery solution is preferably a DNA-free cell recovery solution.

<Example 1>
Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to this embodiment.

(Collection process)
After adding 10.5 mg of sodium salt of EDTA (ethylenediaminetetraacetic acid) as an anticoagulant to 7 mL blood collection tube, 7 mL of peripheral blood was collected from the mother volunteer as volunteer blood after informed consent. I got it. Thereafter, the blood was diluted with physiological saline.

(Concentration process)
Percoll liquid (registered trademark) is used to prepare 1.070 g / mL density liquid and 1.095 g / mL liquid density. The centrifuge tube has a density of 1.095 g / mL at the bottom of the centrifuge tube. 6 ml was added. After the addition, it was placed at 4 ° C. for 30 minutes and cooled. Thereafter, 2 mL of a liquid having a density of 1.070 g / mL is slowly layered on the liquid having a density of 1.095 g / mL so as not to disturb the interface, and then the above-described medium is put on the medium having a density of 1.070 g / mL. Slowly add 11 mL of the blood diluted in step 1 to the centrifuge tube. Then, centrifugation was performed at 2000 rpm (rotation number per minute) for 20 minutes. Thereafter, the centrifuge tube was taken out, and a fraction deposited between a liquid having a density of 1.070 g / mL and a liquid having a density of 1.090 g / mL was collected using a pipette. The concentration process of the same operation was performed on blood collected from the same mother, and a plurality of types of blood fractions collected with a pipette were prepared. All blood fractions were obtained by the same operation.

(Density adjustment process)
The blood fraction collected in the concentration step was washed with physiological saline, and then centrifuged again, and the cell concentration in the fraction was measured using a cell counter (hemocytometer, fully automatic cell counter, TC20, Bio-Rad). ). After the measurement, using a phosphate buffer solution (PBS), the cell concentration was 0.5 × 10 ⁷ cells, 1.0 × 10 ⁷ cells, 4.0 × 10 ⁷ cells, 8.0 × 10 ⁷ cells, 8.0 × 10, respectively. ^Seven and 1.0 × 10 ⁸ were adjusted. The unadjusted fraction that did not adjust the blood cell concentration was also evaluated.

(Smearing process)
The first slide glass substrate (first substrate) was set in a smearing device, and 2 μL of a blood fraction adjusted to a blood cell concentration of 4.0 × 10 ⁷ cells / mL was dropped onto one end thereof. A second slide glass substrate (second substrate) was set in the apparatus and brought into contact with the first slide glass substrate at an angle of 25 ° with a contact force of 0.5N. By touching the lower surface of the second glass slide substrate to the blood fraction and holding that state for 3 seconds, the blood fraction was spread in a space surrounded by two glass slides by capillary action. . While maintaining the angle between the second slide glass substrate and the first slide glass substrate, place the second slide glass substrate in a direction opposite to the region where the blood fraction of the first slide glass substrate is placed. Then, by sliding at a constant speed of 200 mm / s, a smear substrate in which the blood fraction was smeared uniformly on the first slide glass substrate was produced.

  Similarly, smearing was performed on the slide glass substrate for the fractions whose concentration was adjusted as described above and the fractions whose concentration was not adjusted, thereby preparing a smeared substrate.

  The prepared smeared substrate is dipped in Mei Gulunwand staining solution for 3 minutes, immersed in phosphate buffer solution, washed, and then immersed in Giemsa staining solution (diluted with phosphate buffer solution to a concentration of 3%) for 10 minutes. did. Thereafter, it was washed with pure water and dried. In this way, a plurality of stained glass substrates were produced.

  The average distance between cells of the smeared substrate thus prepared was measured. The distance between the cells is a distance between the centers of the cells by circularly approximating the outer periphery of the cells. In addition, for each concentration adjusted in the concentration adjustment step, 50 cells are selected under an optical microscope, the distance from the cell closest to the cell is measured, and the average of the 50 cells is taken. The average distance between them was determined. The degree of blood cell aggregation was also observed. The results are shown in Table 1.

The smear substrate with the blood cell concentration adjusted to 0.5 × 10 ⁷ cells / mL did not aggregate blood cells on the slide glass, but the distance between the cells increased and the cells were very sparse. When the blood cell concentration was decreased, the number of blood cells present on the slide glass decreased, and the efficiency of searching for nucleated red blood cells decreased. By adjusting the blood cell concentration to 1.0 × 10 ⁷ cells / mL, the distance between cells on the substrate could be shortened, and the efficiency of searching for nucleated red blood cells could be increased. In the sample with the blood cell concentration adjusted to 1.0 × 10 ⁸ , there is a possibility that cell aggregation is observed in some places, the cell nucleus is indistinguishable, and the target cell (nucleated red blood cell) cannot be analyzed. It could be confirmed. The slide glass smeared with the fraction whose concentration was not adjusted had a high blood cell concentration, a high frequency of cell aggregation, and a large number of cells that could not be observed. In the present invention, since nucleated red blood cells which are rare cells are selectively obtained one by one, it is preferable that there is no aggregation, and the blood cell concentration of the fraction before smearing is 8.0 × 10 ⁷ cells / mL or less. It was confirmed that the cell aggregation could be reduced by adjusting to.

  In addition, a smear was prepared in the same manner as described above, using a maternal blood sample collected from another mother. The blood cell concentration in the concentration adjusting step was the blood cell concentration shown in Table 2 below. The results are shown in Table 2.

From Tables 1 and 2, as a pretreatment for smearing the fraction on the slide glass, the blood cell concentration is within the range of 1.0 × 10 ⁷ cells / mL or more and 8.0 × 10 ⁷ cells / mL or less. It was confirmed that was uniformly smeared on the slide glass. Furthermore, if the blood cell concentration is 5.0 × 10 ⁷ cells / mL, a smear without aggregation can be prepared, and it is preferable to adjust the blood cell concentration to 5.0 × 10 ⁷ cells / mL or less. Was confirmed.

<A smear with the concentration adjusted under the same conditions>
Separately, using a blood sample collected from a plurality of mothers of other volunteers, a concentration process was performed in the same manner as described above. The fraction containing the obtained nucleated red blood cell candidate cells was diluted with a phosphate buffer (PBS) at a ratio in which the blood cell concentration was adjusted to 4.0 × 10 ⁷ cells / mL. Thereafter, the slide glass substrate was smeared by the same method as described above to prepare a smear substrate, and the cells on the substrate were observed.

  When each specimen was diluted under certain conditions, the blood cell concentration became non-uniform for each specimen, and the uniformity of cells on the slide glass substrate varied. Since the cells are sparse on the glass slide substrate, the distance between the cells is widened, and there are substrates where the efficiency of the analysis is reduced, or there are cells that cannot be analyzed due to cell aggregation being observed, which hinders analysis of blood cells It was confirmed that smears smeared at a certain distance between cells could not be prepared even if dilution was performed under certain specific conditions.

<Contact force during smearing>
Under the above conditions, a smear was prepared by changing the contact force between the first slide glass substrate and the second slide glass substrate. When the contact force at the time of smearing is 2.5 N or less, the shape of the cells after smearing can be maintained in a normal circular shape, and it can be prevented from becoming a shape stretched parallel to the smearing direction. it can. By maintaining the shape of the cell in a circle, it was possible to determine the target nucleated red blood cell when measuring and discriminating the shape of the nucleus by image analysis, and to identify the cell well. . When the contact force is 0.5 N or more, the smear solution is placed in the target area during the operation of bringing the first glass slide substrate and the second glass slide substrate into contact with each other and spreading the fraction (blood) by capillary action. It spreads well, prevents exudation outside the target area, and smears the cells uniformly. In addition, the second slide glass substrate could be stably moved, and a specimen in which cells were smeared uniformly could be produced.

<Movement speed of the second slide glass substrate>
Moreover, the smear board | substrate was produced on the said conditions by changing the speed which moves the 2nd slide glass board | substrate on the 1st slide glass board | substrate. When the moving speed of the second glass slide substrate is 300 mm / s or less, the fraction (blood) can be uniformly spread on the first glass slide substrate, and the cells are prevented from becoming sparse. It was possible to smear uniformly. Further, if it is 0.5 mm / s or more, it prevents the blood cells from being pulled to the second slide glass substrate side, and the phenomenon that the blood cell components are smeared in the latter half of the smear area is eliminated. It was found that a uniform smear was obtained.

  If blood cells do not spread uniformly on the first glass slide substrate and aggregation occurs, and there are nucleated red blood cells derived from fetuses, which are rare cells, it is difficult to obtain and analyze nucleated red blood cells derived from the fetus. become. Therefore, the cells are preferably smeared uniformly so that no aggregation occurs. In order to efficiently perform image detection and cell isolation, it is preferable that the cells are smeared uniformly, and the speed at which the second slide glass substrate is moved on the first slide glass substrate is set to 0. It is preferable to set it to 5 mm / s or more and 300 mm / s or less.

(Selection process based on cell shape)
In order to select nucleated red blood cells that are candidates for fetal nucleated red blood cells from the blood after the concentration step that has been adjusted to 4.0 × 10 ⁷ cells / mL and smeared on a glass substrate, An electric two-dimensional stage (hereinafter referred to as XY stage), an objective lens, a measurement system of an optical microscope equipped with a CCD camera, an XY stage control unit, and a vertical direction (hereinafter referred to as Z direction) moving mechanism. A control unit including a Z direction control unit to be controlled, an image input unit, an image processing unit, and an XY position recording unit was prepared. The smeared substrate prepared as described above was placed on an XY stage, focused on a glass substrate, scanned, an image obtained from an optical microscope was taken in, and nucleated red blood cells as target cells were searched for by image analysis.

In the image analysis, cells satisfying the conditions of the following expressions (1) and (2) are detected, a coordinate system composed of two axes orthogonal to the image to be analyzed is set, and one of the two axes is an X axis, two The position of the detected cell in the X direction and the position in the Y direction when the other axis was the Y axis were recorded.
0.25 <(N / C) <1.0 (1)
0.65 <(N / (L × L)) <0.785 (2)

  Here, C is the area of the cytoplasm of the cell for image analysis (area of the region representing the cytoplasm in the image used for analysis), and N is the area of the nucleus of the cell for image analysis (the cell nucleus in the analysis image). L represents the major axis or diameter of the nucleus of the cell to be analyzed (the elliptical major axis when the region representing the nucleus of the cell in the analysis image is approximated to an ellipse, or the nucleus of the cell in the analysis image) The region to be expressed is defined as a circular diameter when approximated to a circular shape), that is, an elliptical major axis or a circular diameter circumscribing a complex-shaped cell nucleus. Nucleated erythrocytes that are candidates for fetal nucleated erythrocytes existing on the slide glass substrate satisfying these (1) and (2) were selected and used as fetal nucleated red blood cell candidates in the next step.

(Process to sort nucleated red blood cells based on optical properties)
The optical characteristics of ten nucleated red blood cells, which are candidates for fetal nucleated red blood cells selected based on the shape information, were analyzed using a microspectroscope. Ten nucleated red blood cells that are candidates for fetal nucleated red blood cells on a slide glass substrate are identified, and one of the nucleated red blood cells has an absorbance 1 of monochromatic light having a wavelength of 460 nm and a wavelength of 560 nm. The absorbance 2 of monochromatic light was measured, and the absorbance ratio (absorbance 1 / absorbance 2) was calculated. Next, five red blood cells without nuclei are selected as reference red blood cells in the short distance from the nucleated red blood cells in the vicinity of the nucleated red blood cells. Similarly, for each reference red blood cell, The ratio of absorbance (absorbance 1 / absorbance 2) was calculated, and the average value was calculated.

  In the same manner, the same calculation was performed for the remaining nine cells of nucleated red blood cells that are candidates for fetal nucleated red blood cells on a glass substrate. From this calculation result, (the ratio of the absorbance of nucleated red blood cells / the average ratio of the absorbance of reference red blood cells) is calculated, and the cells having the largest difference from “1” are determined as fetal nucleated red blood cells. Cell A, and the cell with the smallest difference from “1” was regarded as the nucleated red blood cell derived from the mother and was designated as cell B.

(Cell isolation process)
Cells A and B determined in the above steps were collected using a micromanipulator. The micromanipulator was a dedicated micromanipulator based on the Narishige microinjection system equipped with an Olympus auto stage microscope. The smear sample containing cells A and B determined in the above process is set on the stage of the micromanipulator, and the position coordinates of the acquired cells A and B are read into the automatic stage of the micromanipulator. Moved to acquisition position. After the movement, the micromanipulator was moved under microscopic observation, the cells were taken out, and the obtained cells were sealed in a tube containing 5 μL of DNA-free cell recovery solution.

(Amplification process [DNA amplification process])
Using the single cell WGA kit manufactured by New England Biolabs, the whole genome was amplified using the cells A and B collected using the micromanipulator, and a small amount of DNA in the cells was about 1 million according to the instructions. Amplified twice.

(Determination step [Determination step from maternal or fetal origin])
The whole genome amplification product amplified from each cell is equally divided and one of them is used, and using the genome analyzer Miseq made by ILLUMINA, the C1QTNF9B gene region, PCDH9 gene region, BRCA2 gene region, MTRF1 gene region of chromosome 13 By comparing SNPs (SNP IDs: rs3751355, rs1799955, rs2297555, rs9571740), it was confirmed that cell A and cell B had different SNPs. From this result, it was confirmed that each cell could be obtained from the slide glass one by one.

  Separately, cells C expected to be leukocytes on the slide glass were collected with a micromanipulator, and SNPs were examined in the same manner as cells A and B. As a result, it was confirmed that they coincided with the SNPs of cell B. From the above, it was confirmed that cell A was a fetal nucleated red blood cell, and cell B was a mother-derived nucleated red blood cell.

  As described above, it was found that prenatal diagnosis is possible by obtaining and analyzing fetal nucleated red blood cell DNA.

  DESCRIPTION OF SYMBOLS 12 ... 1st board | substrate, 14 ... Dropping means, 16 ... Fraction, 18 ... 2nd board | substrate

Claims (8)

A concentration step of concentrating nucleated red blood cells in maternal blood by density gradient centrifugation;
A concentration adjusting step for adjusting the blood cell concentration of a fraction of maternal blood in which nucleated red blood cells are concentrated by the concentration step;
A smearing step of smearing the fraction adjusted in the density adjustment step on the first substrate;
A candidate cell identification step of identifying candidate cells of nucleated red blood cells from the fraction smeared on the first substrate;
Obtaining the nucleated red blood cell candidate cells from the first substrate, and
The concentration adjusting step is a method for obtaining nucleated red blood cell candidate cells, wherein the blood cell concentration is adjusted to 1.0 × 10 ⁷ or more and 8.0 × 10 ⁷ or less per 1 mL of the fraction.   2. The nucleated red blood cell according to claim 1, wherein in the smearing step, an amount of smearing the fraction on the first substrate is 1.0 μL or more and 4.0 μL or less with respect to one sheet of the first substrate. A method for obtaining candidate cells. The smearing process includes
Dropping the fraction onto the first substrate;
An angle formed between the first substrate and the second substrate is 10 ° or more and 60 ° or less, and 0.5N or more and 2 in the fraction on the first substrate. . Contacting with a force of 5 N or less;
Holding the second substrate in contact with the first substrate and spreading the fraction to a contact position between the first substrate and the second substrate;
The second substrate is moved on the first substrate at a speed of 0.5 mm / sec or more and 300 mm / sec or less at an angle and a force when the first substrate and the second substrate are brought into contact with each other. The method for obtaining nucleated red blood cell candidate cells according to claim 1 or 2, comprising the step of smearing the fraction on the first substrate. 4. The nucleated red blood cell candidate according to claim 1, wherein the concentration adjusting step adjusts the blood cell concentration to 2.0 × 10 ⁷ or more and 5.0 × 10 ⁷ or less per 1 mL of the fraction. How to get cells.   5. The method for obtaining nucleated red blood cell candidate cells according to claim 1, wherein the candidate cell identification step identifies based on at least one of a cell shape and optical characteristics. A concentration step of concentrating nucleated red blood cells in maternal blood by density gradient centrifugation;
A concentration adjusting step of adjusting a blood cell concentration of a fraction of maternal blood in which nucleated red blood cells are concentrated by the concentration step to 1.0 × 10 ⁷ or more and 8.0 × 10 ⁷ or less per 1 mL of the fraction;
A smearing step of smearing the fraction adjusted in the density adjustment step on the first substrate;
A candidate cell identification step for identifying candidate cells for nucleated red blood cells from the fraction smeared on the first substrate, and a nucleated red blood cell candidate cell smeared substrate for smearing nucleated red blood cell candidate cells,
A nucleated red blood cell candidate cell smear substrate, wherein an average distance between centers of blood cells contained in the fraction smeared on the first substrate is 30 μm or more and 160 μm or less.   7. The nucleated red blood cell according to claim 6, wherein in the smearing step, an amount of smearing the fraction on the first substrate is 1.0 μL or more and 4.0 μL or less with respect to one sheet of the first substrate. Candidate cell smear substrate. The smearing process includes
Dropping the fraction onto the first substrate;
An angle formed between the first substrate and the second substrate is 10 ° or more and 60 ° or less, and 0.5N or more and 2 in the fraction on the first substrate. . Contacting with a force of 5 N or less;
Holding the second substrate in contact with the first substrate and spreading the fraction to a contact position between the first substrate and the second substrate;
The second substrate is moved on the first substrate at a speed of 0.5 mm / sec or more and 300 mm / sec or less at an angle and a force when the first substrate and the second substrate are brought into contact with each other. The nucleated red blood cell candidate cell smearing substrate according to claim 6 or 7, further comprising a step of smearing the fraction on the first substrate.

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