WO2002073190A1

WO2002073190A1 - Method of sorting particulates with different specific gravities

Info

Publication number: WO2002073190A1
Application number: PCT/JP2002/002317
Authority: WO
Inventors: Hajime Ogata
Original assignee: Hajime Ogata
Priority date: 2001-03-13
Filing date: 2002-03-12
Publication date: 2002-09-19
Also published as: JP2005098704A

Abstract

A method of easily and accurately sorting the groups of particulates containing two or more types of particulates with different specific gravities into two groups, i.e., a high specific gravity group and a low specific gravity group, comprising the step of centrifugally separating the particulate groups together with solid inclusions having a specific gravity positioned in the intermediate zone of the specific gravity of the particulate groups, vertically movable while inscribing in a centrifugal tube, and having an upper part from a portion inscribing in the centrifugal tube formed in projected shape to move high specific gravity particulates to the lower side of the solid inclusions through the tangential line of the centrifugal tube inside wall to the solid inclusions or move low specific gravity particulates to the upper side of the solid inclusions and, at the same time, move the solid inclusions to the intermediate position of two high and low specific gravity groups of particulates, whereby the method can be utilized favorably for separating leucocytes from blood.

Description

Description Separation method of fine particles with different specific gravity

The present invention relates to a method and an apparatus for separating a mixture of two or more types of fine particles having different specific gravities into two groups of light and heavy weights simply and with high accuracy. In particular, the present invention relates to a method and apparatus suitable for removing erythrocytes from blood and separating and collecting nucleated cells such as leukocytes. Background art

Conventional methods for removing erythrocytes from body fluids such as blood to separate and collect nucleated cells include (1) a buffy coat-coating method, (2) a red blood cell removal method by hemolysis, and (3) a hemagglutination sedimentation. Separation method, (4) Centrifugation method using density gradient of specific gravity solution, (5) Separation method of high specific gravity using partition wall fixed in centrifuge tube, (6) Floating solid inclusion for centrifugation Techniques such as a centrifugal separation method using a method are known.

Each of the known methods has the following problems.

(1) Buffy's coat collection method

In this method, it is inevitable that a large amount of red blood cells is mixed into the nucleated cells to be collected, and the recovery rate of the nucleated cells largely depends on the skill of the person performing the procedure.

(2) Erythrocyte removal by hemolysis

In this method, the use of cytotoxic reagents used for hemolysis is inevitable, and the recovery and purity of nucleated cells depend on conditions such as the reagent concentration, the mixing ratio, and the reaction time. Moreover, it is not suitable for processing a large amount of sample.

(3) Hemagglutination sedimentation method

In order to obtain a satisfactory recovery by this method, it is necessary to dilute the sample with a large amount of isotonic solution and repeat the coagulation sedimentation procedure several times. Therefore, after fractionation, it is necessary to centrifuge and wash the suspended cells again to remove flocculants such as dextran and hestastarch, and to collect nucleated cells present in a state of being dispersed in a large amount of solution. It is complicated. (4) Centrifugation using density gradient of specific gravity solution

This method requires careful operation and skill in separating the solution and the target specific gravity band. In addition, it is necessary to centrifuge and wash the suspended cells again after the fractionation to remove a specific gravity solution such as Ficoll and Percoll.

(5) Method for removing high-density fractions containing red blood cells using a septum fixed in a centrifuge tube This method involves placing a specific gravity solution under the septum fixed in a centrifuge tube, and overlaying or centrifuging a sample such as blood. This facilitates the subsequent recovery operation.However, since the specific gravity solution having a volume equal to the volume of the high specific gravity fraction removed after centrifugation moves onto the partition wall, the specific gravity solution is removed as in (4). It is necessary to remove it, and the work process is many and complicated.

(6) Centrifugation method using floating solid inclusion for centrifugation

This method is a method performed by the present inventor and already published as Japanese Patent Application Laid-Open No. Hei 9-155552, the purpose of which is to centrifuge together two or more types of particles having different specific gravities. A solid inclusion that intervenes in the specific gravity band, can move up and down in the centrifuge tube by centripetal force to separate particles into two groups of light and heavy, and has a concave or sloped upper surface whose size just fits the inner wall of the centrifuge tube. It is used. Such a solid inclusion has a large contact area with the wall of the centrifuge tube, and a centrifugal force is applied obliquely outward to the solid inclusion against the inner wall of the centrifuge tube during centrifugation. The higher the centrifugal force, the more difficult it is for solid inclusions to move. Therefore, with the shape specifically described there, the centrifugal force and specific gravity setting conditions for intervening between the erythrocyte layer and the nucleated cell layer after centrifugation are narrow, and further improvements are expected. It is rare.

Also, a method of separating blood cells and the like using a sol-like inclusion for serum separation is already known, but strict specific gravity adjustment of the inclusion is required for use for cell separation. Stable results cannot be obtained because they are necessary and are affected by individual differences in the specific gravity distribution of specimens. Disclosure of the invention

The present inventor has found that a fine particle group containing two or more kinds of fine particles having different specific gravities and a solid inclusion having a specific specific gravity and a shape are centrifuged together, so that the method is more efficient and more efficient than the conventional method. In a simple and convenient way, we found a method for separating fine particles with different specific gravities into two groups with light inclusions in Sakai.

That is, the present invention

(1) A fine particle group containing two or more types of fine particles with different specific gravities has a specific gravity located in the middle zone of the specific gravity of the fine particle group, and can move up and down while inscribed in the centrifuge tube. Centrifuge with the solid inclusions whose upper part is more convex than the contacting part, and pass the tangent line between the inner wall of the centrifuge tube and the solid inclusions to move high specific gravity fine particles below the solid inclusions or low specific gravity. A method for separating fine particles having different specific gravities, wherein the fine particles are moved above the solid inclusions and at the same time, the solid inclusions are moved and interposed between the high and low density two particle groups,

(2) The method according to (1), wherein the solid inclusion is formed into a shape that is in contact with the inner wall of the centrifuge tube by a line.

(3) The method according to (1) or (2), wherein the shape of the solid inclusion is a true sphere, an ellipsoid, a football, a raindrop, a spindle or an abacus.

(4) The method according to any one of (1) to (3), wherein the fine particles have a particle size in the range of 0.01 to 200 jitm.

(5) The method according to any one of (1) to (4), wherein the fine particles are suspended in a liquid or exist as a colloid.

(6) The method according to any one of (1) to (5), wherein the liquid containing fine particles is a body fluid.

(7) The method according to any one of (1) to (6), wherein the solid inclusion is agar, gelatin, synthetic resin, ceramitas, protein or polysaccharide.

(8) The method according to any one of (1) to (7), wherein the centrifuge tube is made of glass or synthetic resin, and

(9) It is made of solid inclusions that can move up and down inside the centrifuge tube while being inscribed in the centrifuge tube, and the upper part of the portion inscribed in the centrifuge tube is formed in a convex shape. 1) an apparatus for performing the method according to any of (8),

Ah .

In the present invention, two or more kinds of fine particles having different specific gravities to be separated are fine particles. Particles having a diameter of about 0.01 to 200 / zm and containing two or more kinds of fine particles with different specific gravities regardless of the type and shape of the fine particles, especially fine particles are mixed in the liquid medium It is a fine particle group in a state. A typical example is a body fluid, particularly blood containing cells having various specific gravities such as red blood cells and white blood cells. Other examples include colloidal liquids containing colloidal particles having different specific gravities, liquids containing micelles, emulsions, suspensions, and the like. In the present invention, it is particularly suitable for collecting nucleated cells by separating and removing red blood cells from a cell population in a body fluid.

The material, size and the like of the centrifuge tube used in the present invention are not particularly limited, as long as they are used in an ordinary centrifuge. That is, it is preferable that the bottom 6 has a shape such as a hemispherical shape, a semiellipsoidal shape, or a cone shape, and a cylindrical body following the bottom 6. The material of the centrifuge tube should be a material that has a gap that allows the particles to move between the wall of the centrifuge tube and the solid inclusion inscribed when a centrifugal force is applied, or such a gap. What has elasticity enough to produce Examples of the hard material include glass and metal materials, and examples of the material having elasticity include synthetic resin and rubber. Among them, those made of glass or synthetic resin such as polypropylene and chloride chloride are preferably used. The inner wall of the centrifuge tube may be coated with a lubricant such as paraffin so that solid inclusions can easily move up and down the centrifuge tube during centrifugation.

The solid inclusion used in the present invention is, when two or more types of fine particles having different specific gravities are dispersed in a liquid, insoluble in the liquid, and when a centrifugal force is applied, the fine particles are in contact with the centrifuge tube wall. A material having a gap that can move between the solid inclusions inscribed therein and a material having flexibility enough to create such a gap, that is, an elastic body is preferable.

Examples of the material of the solid inclusion 2 include agar, gelatin, a silicone resin, a polystyrene resin, a synthetic resin such as an ABS resin and a polyurethane resin, ceramics such as glass, proteins, and polysaccharides. A cavity may exist inside the solid inclusion, and the cavity may be filled with another substance.

One of the features of the present invention is that the shape of the solid inclusions present at the interface of the fine particles having different specific gravities by centrifugation becomes convex above the part inscribed in the centrifuge tube. It was molded as follows.

Specific shapes of the solid inclusion include those formed in a line with the inner wall of the centrifuge tube, such as a true sphere, an oval sphere, a foot pole, a raindrop, a spindle, and an abacus. Among them, particularly preferred is a true sphere or ellipsoid (the ratio of the major axis to the minor axis is preferably within 1: 2).

The specific gravity of the solid inclusions can be appropriately selected depending on the purpose of separating fine particles.For example, a substance having a specific gravity that falls between the specific gravities of two fine particles to be separated, for example, silica, clay The specific gravity can be adjusted arbitrarily by mixing the required amount of ceramic powder, metal powder, etc., making the interior hollow, and enclosing other substances in the cavity.

Next, a typical embodiment of the present invention will be described with reference to the drawings. First, as shown in FIG. 3, a spherical solid inclusion 2 is placed in a centrifugal tube 1 having a hemispherical bottom 6 and a cylindrical body 7 following the same so as to inscribe the centrifugal tube 1. Blood 10 containing two kinds of microparticles having different specific gravities of red blood cells 3 (high-density microparticles) and nucleated cells 4 (low-density microparticles) is placed in this centrifuge tube 1 (Fig. 4), and the centrifuge tube is centrifuged. . At the start of centrifugation, red blood cells 3 having a high specific gravity gather at the part 5 where the inclined surface of the solid inclusion and the inner wall of the centrifugal tube are asymptotic, and when the centrifugal force is further applied, the red blood cells 3 The solid inclusions 2 move downward through the solid inclusions 5 while being pushed up by the negative centrifugal force generated by the specific gravity difference from the red blood cells. At the end of the centrifugation, most of the red blood cells 3 move below the solid inclusions, and the solid inclusions 2 intervene at the interface 9 where the nucleated cells 4 and the red blood cells 3 are in contact (FIG. 6).

A mixture of two or more types of fine particles having different specific gravities, for example, blood, may be arranged below the solid inclusion. In this case, first, a mixture of two or more kinds of fine particles having different specific gravities is put into a centrifuge tube, and a solid inclusion formed thereon is introduced. In this case, the shape of the solid inclusion is preferably spherical or elliptical because of its easy insertion. When a centrifuge tube containing a mixture of two or more types of fine particles having different specific gravities and solid inclusions is centrifuged, the low-density particles move above the solid inclusions through the contact line between the inner wall of the centrifuge tube and the solid inclusions. High specific gravity particles stay below solid inclusions. At that time, the solid inclusions move downward by the amount of the low specific gravity particles moving upward. Centrifuge place The centrifugal force and the time may be appropriately determined depending on the type and amount of the fine particles to be separated.

In the final steady state, the microparticles antagonize the narrow space 5 formed by the asymptotics between the surface 8 of the solid inclusion and the inner wall of the centrifuge tube while leaving a small specific gravity gradient. Even if there is a lot difference, the desired low specific gravity fine particles 4 can be stably separated in a state where other fine particles are not mixed.

After centrifugation, the liquid phase containing the low-specific-gravity particles above the solid inclusions can be easily separated by tilting the centrifuge tube or by using a suction tool such as a pipette to separate the high and low groups of fine particles. it can. BRIEF DESCRIPTION OF THE FIGURES

Figure 1

Plan view of a spherical solid inclusion

Figure 2

Vertical cross section of elliptical spherical solid inclusion

Fig. 3

Vertical sectional view of a centrifuge tube with a spherical solid inclusion

Fig. 4

Schematic diagram of a vertical cross section of a centrifuge tube containing a spherical solid inclusion

Fig 5

Fig. 6

Schematic drawing of the vertical cross section of a centrifuge tube containing the inclined solid spherical inclusions at the end of centrifugation

1. Centrifuge tube

2. Solid inclusions

3. Red blood cells

4. Nucleated cells (white blood cells) 5. Part where the spherical inclusions and the inner wall of the centrifuge tube are asymptotic

6. Centrifuge tube bottom

7. Centrifuge tube body

8. Surface of solid inclusions in contact with fine particles

9. Line where low specific gravity particles and high specific gravity particles touch

10. BEST MODE FOR CARRYING OUT THE INVENTION

Example

Example 1

A polystyrene sphere having a diameter of 10.6 Oram (specific gravity: 1.05) was molded and inserted into a glass tube centrifuge tube having an inner diameter of 10.75 mm and a length of 75 mm. 4 ml of human peripheral blood (specific gravity: about 1.056) was poured into the centrifuge tube, and the mixture was centrifuged at 50 OG for 20 minutes in a centrifuge. Erythrocytes (specific gravity: 1.0964) that settled on the inner tangent to the solid inclusions moved down the solid inclusions along the inner wall of the centrifuge tube, and simultaneously the solid inclusions were pushed upward. Eventually, a fraction containing serum and leukocytes remained above the solid inclusions, and red blood cells accumulated below.

Table 1 summarizes the results of this experiment.

Example 2

Using a polystyrene resin elliptical sphere (short axis to long axis ratio ヽ 1: 1.2, specific gravity 1.05) in contact with the inner wall of the solid inclusion centrifuge tube, centrifuged at 600 G for 15 minutes. Blood was processed as in. The results are also summarized in [Table 1].

Example 1 Example 2

Heart [J '

Oval oval sphere Centrifugal force GX min 0 500X20 600X 15 Volume (ml) 4 1.8 1.7 Red number (X10 ⁴ / ^ 1) 556 30 50 Blood hemoglobin (g / d 1) ,, 011 · A ¾ %) 52.6 3.4 6.0 White number (/ 1) 6,900 14,000 14,200 Number of blood granulocytes (/ 1) 2,300 4, 100 4,400 Income

Rate Leukocyte <%) 100 91.3 87.5

Industrial applicability

In the method of the present invention, a fine particle group containing two or more types of fine particles having different specific gravities can be converted into two groups with high and low specific gravities with high accuracy by simply performing a conventional centrifugal separation operation using a relatively simple apparatus. Can be fractionated. In particular, this method is very suitable for removing red blood cells from blood and collecting leukocytes, which are nucleated cells.

Claims

The scope of the claims

1. A fine particle group containing two or more types of fine particles with different specific gravities has a specific gravity located in the middle zone of the specific gravity of the fine particle group, and can move up and down while inscribed in a centrifuge tube. Centrifuged together with the solid inclusions whose upper part is convex, and passed through the tangent line between the inner wall of the centrifuge tube and the solid inclusions to move the high specific gravity fine particles below the solid inclusions or low specific gravity fine particles. A method for separating fine particles having different specific gravities, wherein the particles are moved above the solid inclusions and at the same time, the solid inclusions are moved and interposed between the two groups of high and low specific gravity particles.

2. The method according to claim 1, wherein the shape of the solid inclusion is shaped to be in contact with the inner wall of the centrifuge tube by a line.

3. The method according to claim 1, wherein the shape of the solid inclusion is a true sphere, an ellipsoid, a football, a raindrop, a spindle, or an abacus.

4. The method according to any one of claims 1 to 3, wherein the fine particles have a particle size in the range of 0.01 to 200 μπι.

5. The method according to any one of claims 1 to 4, wherein the fine particles are suspended in a liquid or exist as a colloid.

6. The method according to any one of claims 1 to 5, wherein the liquid containing fine particles is a body fluid.

7. The method according to any one of claims 1 to 6, wherein the solid inclusion is agar, gelatin, synthetic resin, ceramitas, protein or polysaccharide.

8. The method according to any one of claims 1 to 7, wherein the centrifuge tube is made of glass or synthetic resin.

9. A solid inclusion that is movable up and down inside the centrifuge tube while being inscribed in the centrifuge tube, and formed in a convex shape above the part inscribed in the centrifuge tube. An apparatus for performing the method according to any one of the ranges 1. to 8.