JP2000117150A - Centrifugal separation method and centrifugal container used therefor - Google Patents

Abstract

(57) [Summary] [PROBLEMS] To provide a centrifugation method capable of extracting components required for analysis in a short time. A method of separating a mixture containing a low specific gravity component and a high specific gravity component by centrifugal force, wherein the rotational body has a center of rotation of a circular motion that generates centrifugal force and has a center of rotation. The above mixture is put into a centrifugal container provided with a ring-shaped dam not higher than the outer wall and centrifuged. It is characterized by the following.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a centrifugal separation method and a centrifugal container used therefor, and is particularly preferably used for automatically analyzing centrifugally separated low specific gravity components, for example, serum and plasma in blood in a short time. Can be done.

[0002]

2. Description of the Related Art In general, in blood analysis, serum and blood cells in blood are centrifuged, the serum is taken out, reacted with a reagent, and the degree of discoloration of the reagent is measured. In addition to blood analysis, centrifuges are widely used to extract specific components from a fluid containing a plurality of components having different specific gravities.

As shown in FIG. 11, a conventional centrifuge comprises a rotating machine R which is driven to rotate by a motor (not shown) and an even number of centrifuge tubes T whose one end is closed.
Then, with the sample placed in the centrifuge tube T, the centrifuge tubes are loaded into the rotating machine R so as to be equally spaced in the circumferential direction. When the sample is small, the sample is put in one centrifuge tube T, and a fluid having the same weight as the sample is put in another centrifuge tube as a dummy.
Are mounted so as to be located at diagonal positions of the rotating machine R with respect to each other.

[0004]

Problems to be solved by the above-described conventional centrifugal separator have many problems as follows. First, the upper layer components in the centrifuge tube cannot be removed until the rotating body R stops.

Second, when the upper layer component in the centrifuge tube is to be automatically collected by a sampling device such as a suction pipette of the analyzer, the stop position of the centrifuge tube is detected, and based on the detection signal. Either the sampling device has to be moved or the centrifuge tube has to be forcibly stopped at the position where the sampling device has been moved, in any case a complicated control circuit was required. Third, because the centrifuge itself is large,
The analyzer cannot be downsized.

[0006] Therefore, a first object of the present invention is to provide a centrifugation method capable of extracting components required for analysis in a short time. A second object is to make it possible to automatically collect an upper layer component with a sampling instrument of an analyzer without requiring a complicated control circuit. A third object is to reduce the size of the centrifuge.

[0007]

According to the present invention, there is provided a centrifugal separation method according to the present invention, wherein a centrifugal force is generated in a method for separating a mixture containing a low specific gravity component and a high specific gravity component by centrifugal force. The mixture is centrifuged in a centrifugal container provided with a ring-shaped dam centered on the center of circular motion as the center of rotation and having a ring-shaped dam centered on the center of rotation and not higher than the outer wall, and centrifuged. It is characterized in that the high specific gravity component is dammed by the dam and the low specific gravity component is stored at the position of the rotation center.

According to the present invention, since a ring-shaped dam which is not higher than the outer wall is provided inside the centrifugal container, when the mixture is put at the center of rotation and rotated, the high specific gravity component is reduced by the centrifugal force. Overcoming the dam and colliding with the outer wall over the components. Thereafter, the high specific gravity component sinks between the dam and the outer wall as the rotation speed decreases. On the other hand, the low specific gravity component flows over the dam and flows backward toward the center of rotation because it is in the upper layer. Therefore, the centrifuged low specific gravity component is stored at the center of rotation, while the high specific gravity component is blocked by the dam. Therefore, even if a sampling instrument such as a pipette tip is inserted into the low specific gravity component at the center of rotation during rotation, it is difficult to remix with the high specific gravity component. As a result, a low-specific-gravity component can be sampled without waiting for the rotating body to stop, and a complicated control circuit is not required to move the sampling tool.

While the centrifugal separation method of the present invention is being carried out, the number of revolutions of the circular motion causing the centrifugal force is detected, and when the number of revolutions falls below a predetermined value, the sampling instrument is moved to a low specific gravity on the rotating shaft. If the components are immersed in the components, the components can be collected in a short time after the start of centrifugation.

[0010] A centrifugal container suitable for carrying out the centrifugal separation method of the present invention generates a centrifugal force in a container in which a mixture containing a low specific gravity component and a high specific gravity component is separated by centrifugal force. It has a rotating body shape with the center of the circular motion to be rotated as the center of rotation, and a dam not higher than the outer wall is provided inside.

According to this centrifugal container, since the centrifugal container itself is in the form of a rotating body, a centrifugal force can be generated by rotating the center of the container so as to coincide with the centrifugal axis. Therefore, no dummy container is required. Since the dam is provided as described above, during centrifugation, the mixture collides with the dam and collides with the outer wall, but as the rotation speed decreases, the high specific gravity component sinks between the dam and the outer wall. On the other hand, the low specific gravity component passes over the dam in the opposite direction and accumulates at the rotation center position.
Therefore, the low specific gravity component and the high specific gravity component are not easily remixed after centrifugation. Further, since the low specific gravity component accumulates at the rotation center position, the low specific gravity component can be collected from the rotation center.

In the above-mentioned centrifugal container, it is preferable that a pool lower than the dam is provided in a region inside the dam and including the center of rotation in order to store a low specific gravity component. This is because the low specific gravity component can be more reliably prevented from returning to the outside of the dam and being remixed with the high specific gravity component. The dam preferably has an outwardly projecting flange at its upper end, since the flange prevents high specific gravity components from trying to get over the dam.

The centrifuge container of the present invention does not require a lid.
Even if there is no lid, by making the outer wall of the container sufficiently higher than the dam, fluid can be prevented from spilling during centrifugation. However, if a lid is provided to close the opening of the container, the outer wall of the container can be lowered,
The size can be reduced. The lid must have a hole for sampling at the center of rotation.

When the lid is made of a rigid material, the dam preferably has a height such that its upper end does not touch the lid. This is because the gap between the upper end of the dam and the lid can be a flow path where the high specific gravity component goes to the outside or the low specific gravity component goes to the rotation center.

When the lid is made of an elastic material, it is preferably adhered to the outer wall of the container, and the dam has a height such that its upper end is in contact with the lid. The lid is elastically deformed and bulges upward when a centrifugal force is generated because it is adhered to the outer wall of the container, so that a gap is formed with the upper end of the dam. Therefore, the high specific gravity component can get over the dam through the gap. On the other hand, since the lid returns to its original shape and comes into contact with the dam with a decrease in the centrifugal force, remixing of the high specific gravity component outside the dam and the low specific gravity component inside the dam can be more reliably prevented. Because.

[0016]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiment 1 An embodiment of the present invention will be described with reference to the drawings. FIG. 1 is a sectional view in the rotation axis direction showing the centrifugal container of the first embodiment. The centrifugal container 1 includes a main body 2 in the shape of a rotating body having a mouth opened upward,
A lid 3 for closing the opening of the main body 2; The material of the main body 2 and the lid 3 is a resin such as polystyrene, ABS resin, polycarbonate, polypropylene, polyethylene, etc., which can give appropriate rigidity. Therefore, the centrifugal container 1 can be easily manufactured by a known technique such as injection molding or cutting. Inside the main body 2, a columnar dam 4 is provided at a central portion including a rotation center, and a deep moat 5 is formed around the dam 4. The bottom of the moat 5 is flat. The lid 3 has a flat disk shape except that it has a through hole 6 for inserting and removing a sample in the center.

Next, using the centrifugal container 1, two
An operation of centrifuging a mixed solution containing components, for example, blood composed of serum (low specific gravity component) and blood cells (high specific gravity component), and a phenomenon occurring in the centrifugal container 1 at that time will be described.

First, a needle of a syringe (not shown) is inserted through the through hole 6 into the centrifugal container 1 with the lid 3 placed on the main body 2, and the blood BL is transferred to the main body 2 as shown in a sectional view in FIG. Fill inside. The filling amount is such that the liquid level is higher than the dam 4 and does not contact the inner surface of the lid 3. In order to rotate the centrifugal container 1, a rotating machine N for rotating the centrifugal container 1 around the center of rotation as shown in a sectional view in FIG. Then, the centrifugal container 1 filled with the blood BL is fixed to the rotating machine N. The blood BL may be filled after the centrifugal container 1 is fixed to the rotating machine N. In this state, when the rotating machine N is driven to rotate the centrifugal container 1,
Due to the centrifugal force, the blood cells C are separated outside the moat 5 and the serum S is separated inside.

After a lapse of a predetermined time at a predetermined number of revolutions, the driving force of the rotary machine N is cut off to rotate by inertia, or the output of the rotary machine N is gradually reduced. As the rotation speed decreases, the centrifugal force weakens, blood cells C accumulate at the bottom of the moat 5, and serum S accumulates thereon or on the dam 4. Since the height of the dam 4 serves as a barrier, the blood cells C do not rest on the upper surface of the dam 4. Therefore, a suction pipette (not shown) can be inserted into the centrifugal container 1 through the through-hole 6 to suck only the serum S. Also,
Since the position of the through hole 6 is always at the center of rotation even during the rotation of the centrifuge container 1, the serum S can be sucked by inserting the suction pipette into the centrifuge container 1 without waiting for the centrifuge container 1 to stop. Therefore, analysis can be started in a short time after centrifugation.

Embodiment 2 A second embodiment of the present invention will be described with reference to FIG. The centrifugal container 11 of this embodiment has the same shape and the same shape as the centrifugal container 1 of the first embodiment, except that a flange 14a protruding in the radial direction is formed at the upper end of the dam 14. The flange 14 a makes it difficult for the high specific gravity component to be placed on the upper surface of the dam 14. Therefore, the collar 14
Compared with the centrifugal container 1 of the first embodiment where a is not formed, the centrifugally separated high specific gravity component and low specific gravity component are more difficult to remix.

Embodiment 3 A third embodiment of the present invention will be described with reference to FIG. In the centrifugal container 21 of this embodiment, a pool 27 that is depressed is provided in a region including the rotation center inside the dam 24. The volume of the pool 27 is about the volume of blood that can be collected at one time with a syringe. An annular convex portion 28 is provided on the periphery of the upper surface of the main body 22, and an annular concave portion 29 complementary to the convex portion 28 is provided on the lower surface of the lid 23, and the convex portion 28 and the concave portion 29 are fitted. By fitting, the lid 23 is positioned with respect to the main body 22. In other respects, it is basically the same as the first embodiment.

The operation and phenomenon when centrifuging blood using this centrifugal container 21 are as follows. Lid 23
The centrifuge container 21 is combined with the rotating machine N (FIG. 3).
(See Reference). A predetermined amount of blood is filled in the pool 27 as shown in FIG. 7 by inserting a needle of a syringe into the through hole 26. The rotating machine N is driven to generate a centrifugal force. The separated blood cells C accumulate in the moat 25 and the serum S accumulate in the pool 27 as shown in FIG. 8, and the dam 24 prevents remixing of the two.
Unlike the first embodiment, since the pool 27 is recessed from the upper end of the dam 24, the following two advantageous effects occur.
First, when the serum S is sucked by the suction pipette, the possibility that the serum S is agitated and enters the moat 25 is lower than in the first embodiment. Second, even if the amount of blood to be filled in the centrifuge container 21 is small, the depth of the serum S necessary for immersing the suction pipette in the serum S can be ensured.

Embodiment 4 A fourth embodiment of the present invention will be described with reference to FIG. In the centrifugal container 31 of this embodiment, the cross-sectional area in the horizontal direction decreases as the moat 35 moves toward the bottom, and conversely, as the dam 34 moves toward the upper end. And the bottom of the moat 35 and the dam 3
The upper end of 4 is sharp in an axial sectional view. The height of the dam 34 is the same as the outer wall of the main body 32, unlike the previous embodiments. Further, the lid 33 is different in material from the previous embodiments, and is made of a thin film. The lid 33 is always in contact with the upper end of the dam 34, and its periphery is fixed to the upper surface of the periphery of the main body 32. Known techniques such as heat fusion and ultrasonic fusion can be applied to the fixing means.

The operation for centrifuging blood using the centrifugal container 31 of this embodiment is the same as that of the third embodiment, but the phenomenon is slightly different as follows. When blood is filled into the pool 37 by passing the needle of the syringe through the through hole 36 and the centrifugal container 31 is rotated, the lid 33 expands upward as indicated by a virtual line by centrifugal force. Accordingly, a gap is formed between the lower surface of the lid 33 and the upper end of the dam 34, through which blood cells pass toward the moat 35. After the centrifugation, the centrifugal force decreases as the rotation speed decreases, and the lid 33 returns to its original shape. As a result, the lower surface of the lid 33 contacts the upper end of the dam 34 again, and the moat 35 is closed. Therefore, the blood cells accumulated in the moat 35 and the serum accumulated in the pool 37 do not mix again. Further, the centrifugal container 31 of this embodiment has an advantage that it is easy to mold since the moat 35 has a smaller area toward the bottom.

Embodiment 5 A fifth embodiment of the present invention will be described with reference to FIG. This embodiment is a configuration diagram showing a system for automatically collecting a low-specific-gravity component centrifuged. The centrifugal container 41 of the present invention is set to be rotatable around the center of the centrifugal container by a motor M driven by a DC power supply DC. As the centrifugal container 41, any of the above-described first to fourth embodiments may be used, and other shapes may be used as long as they belong to the scope of the present invention.

In the above state, the sample is dropped and filled into the centrifugal container 41 with an unillustrated syringe from which an analysis sample such as blood is collected. Then, the motor M is driven and the centrifugal container 41 is driven.
To rotate. During rotation, the rotation speed is detected by the rotation speed detection sensor PI, and is displayed on the oscilloscope Q.
The signal is transmitted to the control device 50, and the control device 50 cuts off the power supply DC after a certain time from when the rotation speed reaches a predetermined high value. The motor M rotates for a while with the centrifugal container 41 due to inertia. When the rotation speed reaches a predetermined low value, the control device 50 lowers the pipette 60 to the center of the centrifugal container 41 and sucks the centrifuged low specific gravity component. The position of the pipette 60 may be controlled only in the vertical direction so as not to hit the bottom of the centrifuge container 41.

[0027]

EXAMPLES-Example 1-This is an experimental example in which blood was centrifuged using the centrifugal container of the first embodiment. In the centrifuge container 1 shown in FIG. 1, the inner diameter of the main body 22 is 14 mm, the depth of the moat 5 is 4 mm,
The height of the dam 4 was 2 mm, and the outer diameter of the dam 4 was 6 mm.

Then, the main body 22 is filled with 560 μl of blood having a hematocrit value of 46, and the lid 3 is covered thereon.
Rotated at 10,000 rpm for 3 minutes at room temperature. At this time, the output of the motor serving as the rotation drive source was 1.3 V. The centrifugal force RCF at this time was calculated as 783 G from the following formula: RCF = 11.18 × 10 ⁻⁶ × r × N ² r: centrifugal radius (unit: cm) N: rotation speed (unit: rpm) Subsequently, the output of the motor is set to 0.
The rate was lowered at a rate of 1 V / sec, and when the voltage dropped to 0.3 V, the rate was lowered at a rate of 0.05 V / sec. Finally, the centrifugal container 1 was stopped. As a result, most of the blood cells accumulated in the moat 5. Then, a pipette is inserted through the through-hole 6 and the dam 4 is inserted.
The serum collected on the upper surface of the sample could be collected.

Example 2 This is an experimental example in which blood was centrifuged using the centrifugal container of the third embodiment. In the centrifugal container 21 shown in FIG. 6, the inner diameter of the main body 22 is 14 mm, and the depth of the moat 25 is 2 m.
m, the depth of the pool 27 from the lower surface of the lid 23 was 5 mm, and the inner diameter of the upper edge of the pool 27 was 9 mm. The gap t between the lower surface of the lid 23 and the upper end of the dam 24 was changed according to the height of the dam 24.

Then, the pool 27 of the centrifugal container 21 was filled with 220 μl of blood having a hematocrit value of 46.3, and
The motor was turned off at 000 rpm for 1 minute and the motor was turned off. The centrifugal force was calculated as 503G by substituting r = 4.5 into the calculation formula of Example 1. As a result, as shown in Table 1, the separation performance was different depending on the value of t.

[0031]

Table 1----------------------------t-value (mm) Results-------------------------------------------------------- −−−−−−−−− 0.5 After stopping rotation, blood cells and serum were mixed. 0.2 After the rotation was stopped, blood cells and serum were mixed. After stopping the rotation, serum could be collected. −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− (−) If so, it is clear that serum can be collected after cessation.

The serum collected at t = 0.1 was 50
μl. Therefore, the separation performance of this example was compared with that of a conventional centrifugal separator, and the results are shown in Table 2. As a conventional centrifuge for comparison, a centrifuge CF-9510 manufactured by Kyoto Daiichi Kagaku Co., Ltd. was used. In addition, CF
The centrifugal force of -9510 was 1500G. The blood cell amount for the collected serum in the table is a value measured with a microscope.

[0033]

Table 2-------------------------------------------------------------------------------Centrifuge −−−− Example 2 (t = 0.1) 1/5000 CF-9510 1/10000 −−−−−−−−−−−−−−−−−−−−−−−−−− According to Table 2, the comparative example Although the blood cell volume is smaller and seemingly superior, the centrifugal force of the present example is 503 G, while the centrifugal force of the comparative example is 1500 G. Clearly, the performance is never inferior. Moreover, in the case of the present embodiment, since the serum can be collected from the rotation center position, the position control of the pipette is easy. In contrast, the comparative example is disadvantageous in that the stop position of the centrifuge tube must be detected and the pipette must be moved to that position. Also, in the case of the comparative example, the centrifuge is large because the dummy centrifuge tube must be placed at a diagonal position. In this regard, the present embodiment is excellent because a centrifugal container for a dummy is not required.

[0034]

As described above, according to the centrifugal separation method and the centrifugal container of the present invention, the low specific gravity component accumulates at the center of rotation, so that the low specific gravity component can be collected without waiting for the rotating body to stop. . Therefore, a low specific gravity component required for analysis can be collected in a short time. In addition, the upper layer component can be automatically collected by a sampling instrument of the analyzer without requiring a complicated control circuit. And a centrifuge can be miniaturized.

[Brief description of the drawings]

FIG. 1 is a sectional view in the rotation axis direction showing a centrifugal container of a first embodiment.

FIG. 2 is a sectional view showing a state in which blood is filled in the centrifugal container of FIG.

FIG. 3 is a cross-sectional view showing a state where the centrifugal container in the state of FIG. 2 is being rotated.

FIG. 4 is a cross-sectional view showing a state where the number of revolutions has been reduced or stopped from the state shown in FIG. 3;

FIG. 5 is a sectional view in the rotation axis direction showing a centrifugal container of a second embodiment.

FIG. 6 is a sectional view in the rotation axis direction showing a centrifugal container according to a third embodiment.

FIG. 7 is a cross-sectional view showing a state in which blood is filled in the centrifuge container of FIG.

FIG. 8 is a sectional view showing a state after centrifugation from the state of FIG. 7;

FIG. 9 is a sectional view in the rotation axis direction showing a centrifugal container of a fourth embodiment.

FIG. 10 is a configuration diagram illustrating a system according to an embodiment of a centrifugal separation method.

FIG. 11 is a conceptual diagram showing a conventional centrifuge.

[Explanation of symbols]

 1,11,21,31,41 Centrifuge container 2,12,22,32 Main body 3,13,23,33 Lid 4,14,24,34 Dam 5,15,25,35 Moat 6,16,26,36 Through hole 27, 37 Pool

Claims (8)

[Claims] 1. A method for separating a mixture containing a low specific gravity component and a high specific gravity component by centrifugal force, comprising: a rotating body having a center of rotation of a circular motion generating a centrifugal force; The above mixture is put into a centrifugal container provided with a ring-shaped dam that is not higher than the outer wall at the center and centrifuged, and the centrifuged high specific gravity component is dammed by the dam, and the low specific gravity component is placed at the position of the rotation center. A centrifugal separation method characterized by storing. 2. The method according to claim 1, wherein during the centrifugal separation method according to claim 1, the number of rotations of the circular motion causing the centrifugal force is detected,
A low specific gravity component collecting method, characterized in that when the number of rotations is equal to or less than a predetermined value, a sampling tool is immersed in a low specific gravity component on a rotating shaft and collected. 3. When a mixture containing a low-specific-gravity component and a high-specific-gravity component is separated by centrifugal force, a container for containing the mixture has a rotating body shape having a center of rotation of a circular motion generating centrifugal force as a center of rotation. And a dam that is not higher than the outer wall is provided inside. 4. The centrifuge container according to claim 3, wherein a pool lower than the dam is provided in a region inside the dam and including the center of rotation to store a low specific gravity component. 5. The centrifugal container according to claim 3, wherein the dam has a flange protruding outward at an upper end thereof. 6. The centrifugal container according to claim 3, further comprising a lid for closing an opening of the container except for having a sampling hole at the center of rotation. 7. The centrifuge container according to claim 6, wherein the lid is made of a rigid material, and the dam has a height such that an upper end thereof does not touch the lid. 8. The centrifugal container according to claim 6, wherein the lid is made of an elastic material and is adhered to an outer wall of the container, and the dam has a height such that an upper end thereof contacts the lid.