Patent Grant
7939034
1. Field of the Invention
The present invention relates to a method and tool for collecting blood plasma, and particularly to a technology for effectively collecting only blood plasma by separating blood plasma and blood cells (red blood cells, white blood cells, blood platelets) in blood from each other in a short time before a blood test.
2. Description of the Related Art
When a blood test is conducted, blood of several mL is collected depending on measurement items by a vacuum blood collecting tube and the vacuum blood collecting tube is set up on various automatic analyzers. The automatic analyzers are large and some hospitals may sometimes ask a test center to inspect blood because of no possession of test equipment. Therefore, it is the present situation that it takes a time to obtain the inspection result. When an emergency clinic test is conducted, it is required to go to facilities having test equipment, so that the test itself is a physical load to old people and the sick. Further, if the environment allowing a blood test to be conducted more readily and more frequently is prepared, protective measures against illness may be enhanced and the quality of life may be improved.
Now, blood used for a blood test may be frequently analyzed optically, and it is known that a solid body in blood, for example, hemoglobin in red blood cells, especially its own color prevents colorimetric measurement. Therefore, in order to provide an accurate test result, as pretreatment for a blood test, it is necessary to prepare a specimen only composed of blood plasma (including serum) obtained by separating blood cells from blood. For this purpose, in order to readily collect only blood plasma by separating to remove blood cells from blood at the site where a blood test is conducted, not limited to a big hospital or a test center equipped with a large blood plasma collection system, the blood plasma collection system has to be made smaller to be a simple test instrument and a blood plasma collection method is required to be made simpler. Further, to mitigate a physical and psychological impact of a patient undergoing a blood test, it is necessary to test by a small amount of blood collected.
In such circumstances, the following items are required for a blood plasma collection method and tool.
(1) Blood plasma collection has to be simple using a small tool and an operation for collection of a small amount of blood. For example, in order to readily implement collection of blood and to allow test to be conducted by anyone anywhere, an amount of blood collected has to be reduced to the degree of several dozens μL at the most, which may be collected by a lancet.
(2) A time necessary to collect blood plasma, that is, a separation time for separating blood plasma and blood cells from each other has to be shortened as much as possible, because fresh blood changes as time passes and becomes unusable for test.
(3) Medicines such as a separation agent do not have to be used, because they may degrade test accuracy.
For these items, there are the following systems as a conventional blood plasma collection system or tool.
Japanese Patent Application Laid-Open No. 2003-83958 discloses a method for separating blood plasma and blood cells from each other by the centrifugal separation method, and the method has a problem of a long separation time, as well as an additional operation for putting blood plasma into a blood test device after separating out blood cells. Japanese Patent Application Laid-Open No. 2006-52950 discloses improvement in size of a device which, based on the centrifugal separation method, was made large, and proposes a method that blood is introduced into a microchip and the microchip is entirely run by a centrifugal separator, but miniaturization of the device is restricted by necessity of a high speed rotating part.
Japanese Patent Application Laid-Open No. 2003-270239 discloses a method for separating blood plasma and blood cells from each other by the membrane separation method, and the method can collect a very small amount of blood to separate, but to implement this method, dilution of the collected blood or extraction of blood plasma to be tested from a small container is required, resulting in complicated operation. Further, because a membrane, used for the membrane separation method, itself has water holding capacity, a part of a specimen may be readily damaged, moreover because it is necessary to push out blood into a filter by pressurization, unfortunately the filter may clog or hemolysis may occur.
Japanese Patent Application Laid-Open No. 2000-171461 discloses a method except the centrifugal separation method and the membrane separation method, in which, by alternately laying cation exchange material and anion exchange material one on the other on a surface of a substrate for introducing blood, the surface of the substrate is made to be charged with cations, and then red blood cells (a sort of blood cells) charged with anions are captured electrically on the surface of the substrate. However, this method has a problem of troublesome coating, as well as quite conceivable, electrostatic attachment of components in blood plasma.
Further, Japanese Patent Application Laid-Open No. 2005-292092 also discloses a method except the centrifugal separation method and the membrane separation method, in which solid components and liquid components in blood are separated from each other by adding flocculant (separation assistant) to a small amount of blood to generate aggregate and precipitating out the aggregate through a channel structure. However, this method has a problem that the channel has a complex shape and it takes a time to effect agglutination, moreover the flocculent may have a bad influence on a blood test.
Further, conventionally, blood plasma having blood cells separated out is generally inspected at another place, which is a time loss, and also a specimen loss. Therefore, desired is a method by which separation of blood cells and a blood test can be conducted concurrently at the same place.
However, as described above, the conventional blood plasma collection method and device have merits and demerits, and cannot meet the requirements according to the items (1) to (3) described above.
The present invention was made in view of these circumstances, and an object of the present invention is to provide a blood plasma collection method by which blood plasma can be collected by accurately, readily separating blood plasma and blood cells in a small amount of blood from each other in a short time, using a small tool having a simple structure, and the tool.
A first aspect of the present invention, to achieve the object described above, provides a blood plasma collection tool including: a separation part for separating blood cells from blood due to spontaneous sedimentation; a collection part for collecting blood plasma obtained by separating out blood cells in the separation part as supernatant fluid; and an overflow channel for overflowing the blood plasma separated out in the separation part to the collection part, in which the separation part is a long microspace having a depth in the direction of gravitational force of not greater than 1 mm.
According to the first aspect, a phenomenon that blood cells in blood spontaneously precipitate due to an effect of gravitational force takes place in the very narrow microspace (separation part) having the very narrow depth in the direction of gravitational force of not greater than 1 mm, and the overflow channel functions as a dam against the separation part so that the blood plasma separated out as supernatant fluid can overflow beyond the overflow channel, whereby the blood cells separated out can be prevented from entering the collection part. Therefore, blood plasma can be collected by accurately and readily separating blood plasma and blood cells in a small amount of blood from each other in a short time, using a small tool having a simple structure.
A second aspect of the present invention is according to the first aspect, in which let H (m) be the depth of the separation part in the direction of gravitational force, L (m) be a length of the separation part in the flow direction of the blood, and Hb (m) be a sedimentation distance in which the blood cells can precipitate while the blood passes through the length (L) of the separation part, then it is the condition that a height h (m) of a bottom surface of the overflow channel relative to a bottom surface of the separation part is made to meet the following items (A) or (B):
(A) When Hb≧H, h≧2×10−5.
(B) When Hb<H, h≧H−Hb.
According to the second aspect, the height h of the bottom surface of the overflow channel relative to the bottom surface of the separation part is set to meet the conditions (A), (B), so that blood cells separated out due to spontaneous sedimentation can be prevented from entering the collection part, and therefore, blood plasma can be collected accurately.
A third aspect of the present invention is according to the first or second aspect, in which a dam is provided in the overflow channel.
According to the third aspect, blood cells which could not be separated out in the separation part can be separated out before the collection part by the dam provided in the overflow channel, and therefore blood plasma can be collected with small contamination of blood cells.
A fourth aspect of the present invention is according to any one of the first to third aspects, in which a filter is provided in the overflow channel.
According to the fourth aspect, blood cells which could not be separated out even in the separation part can be unfailingly separated out before the collection part by the filter provided in the overflow channel, and therefore blood plasma can be collected with very small contamination of blood cells. Further, blood from which most of blood cells were separated in the separation part is passed through the filter, and therefore it is difficult for the filter to be clogged.
A fifth aspect of the present invention is according to any one of the first to fourth aspects, in which a plurality of the separation parts are provided in series.
According to the fifth aspect, blood cells which could not be separated even in the separation part can be unfailingly separated before the collection part, and therefore blood plasma can be collected with small contamination of blood cells.
A sixth aspect of the present invention is according to any one of the first to fifth aspects, in which an inlet of the separation part is formed in the central part of a side wall face of the separation part in the depth direction.
According to the sixth aspect, a distance between an interface of blood introduced into the separation part and an upper surface of the separation part becomes equal to a distance between the interface of blood and a bottom surface of the separation part. Accordingly, when blood is introduced into the separation part, the blood can be prevented from covering only one of the upper surface and the bottom surface of the separation part and spreading. Therefore, it becomes easy to push out blood in the separation part to flow, whereby accurate separation of blood plasma from blood can be provided.
A seventh aspect of the present invention is according to any one of the first to sixth aspects, in which a cross section shape of a bottom of the separation part on a cross section in the width direction of the blood flow is of V-shaped type whose depth increases from both ends toward the central portion.
According to the seventh aspect, blood cells separated out can be stably captured, and therefore the blood cells can be prevented from reentering blood plasma.
An eighth aspect of the present invention is according to any one of the first to seventh aspects, in which side wall faces of the separation part on the side of the inlet and (or) an outlet on a cross section along the flow direction of the blood are inclined to the direction of gravitational force and the angle of inclination is smaller than 90°.
According to the eighth aspect, the side wall faces of the separation part on the side of the inlet and (or) the outlet are inclined to the direction of gravitational force, and therefore a separation rate of blood cells can be enhanced by a boycott effect. Moreover, blood cells can be prevented from entering the collection part of blood plasma by generating a flow in the direction opposite to the direction of the blood flow.
A ninth aspect of the present invention, to achieve the object described above, provides a blood plasma collection method including the steps of: separating blood cells from blood due to spontaneous sedimentation; and collecting blood plasma obtained as supernatant fluid by separating out blood cells in the step of separating, in which the blood is made to flow in laminar flow from the step of separating to the step of collecting.
A tenth aspect of the present invention is according to the ninth aspect, in which the blood cells are separated out by making the blood to flow in a microspace having a depth in the direction of gravitational force of not greater than 1 mm in the step of separating.
According to the present invention, blood plasma can be collected by accurately, readily separating blood plasma and blood cells in a small amount of blood from each other in a short time using a small tool having a simple structure.
Now, preferred embodiments of a blood plasma collection method and tool according to the present invention will be hereinafter described with reference to the accompanying drawings.
First, one example of an embodiment according to the present invention will be described. A blood plasma collection method of the present embodiment is a method for separating blood plasma and blood cells from each other due to spontaneous sedimentation in a short time, using a blood plasma collection tool in which a very narrow microspace having a depth in the direction of gravitational force of not greater than 1 mm is formed. In addition, blood used for the present invention is not limited to human blood, but may be blood of various animals.
As shown in
The separation part 14A is a narrow, long microspace having a depth in the direction of gravitational force of not greater than 1 mm. Further, on the downstream side of the separation part 14A, the collection part 16A is formed. Then, a downstream end of the separation part 14A and an upstream end of the collection part 16A are communicated with each other by the overflow channel 18A. This overflow channel 18A functions as a dam for partially isolating the collection part 16A from the separation part 14A.
Further, an upstream end of the separation part 14A is in communication with a flow channel 20A in communication with a fluid storage 24 which is a columnar, hollow portion formed in the cover plate 22. Further, in a part of the cover plate 22 corresponding to the collection part 16A, a collection port 28 for externally collecting blood plasma collected in the collection part 16A and an air vent 30 for communicating the inside of the collection part 16A with the outside air are formed. The collection port 28 is adapted to fit to a tight seal member 28A.
As shown in
A width W of the separation part 14A in the horizontal direction (see
A length L of the separation part 14A in the flow direction is preferably not smaller than 1 mm and not greater than 200 mm, more preferably not smaller than 1 mm and not greater than 50 mm, and further more preferably not smaller than 1 mm and not greater than 25 mm, taking into consideration easiness of handling of the blood plasma collection tool.
A volume of the separation part 14A is set so that there is enough room to contain blood cells which precipitated due to separation without blocking of the flow channel. A hematocrit value of blood (ratio of a volume of red blood cells contained in a constant amount of blood), though there are individual differences, is approximately to the degree from 33 to 55%. If the volume of the separation part 14A is smaller than an amount of blood to be processed (blood throughput), the volume of the separation part 14A is set to be not smaller than 33 to 55% of the blood throughput. Further, the volume of the separation part 14A may be set to be a volume able to contain the blood throughput. Therefore, the volume of the separation part 14A is preferably not smaller than 0.5 μL and not greater than 50 μL, and more preferably not smaller than 0.5 μL and not greater than 10 μL.
In order to prevent blood cells which spontaneously precipitated in the separation part 14A from reentering the collection part 16A, a height h from the bottom surface of the separation part 14A to a bottom surface of the overflow channel 18A (hereinafter, simply called “height h of the overflow channel 18A”) is set as follows.
A sedimentation rate vb (m/sec) of blood cells (in the direction of gravitational force) may be expressed by the following expression (1):
vb=(2/9)·{(ρ1−ρ2)·g·r2/η} (1)
{vb: sedimentation rate of blood cells (m/sec), ρ1: density of blood cells (kg/m3), ρ2: density of fluid (blood plasma) (kg/m3), g: gravitational acceleration (m/sec2), r: radius, supposing that a blood cell is spherical (m), η: viscosity of fluid (blood plasma) (kg/(m·sec))}
An initial rate vo at which blood flows into the separation part 14A (average flow rate of components in the horizontal direction) may be expressed by the following expression (2), where a supply flow rate of blood is Q (m3/sec):
vo=Q/(H·W) (2)
Therefore, a sedimentation time of blood cells t (sec) in the separation part 14A may be expressed by t=L/vo. Then, the sedimentation distance Hb (m) of blood cells of blood in the separation part 14A may be expressed by the following expression (3):
Hb=vb·L/vo (3)
Then, when Hb≧H, as shown in
On the contrary, when Hb<H, as shown in
Cross section shapes of the separation part 14A, the collection part 16A, the overflow channel 18A and the flow channel 20A taken along the direction of the line B-B are not especially restricted, and various shapes such as a rectangle (square, oblong), a trapezoid, a V shape and a semicircle may be used. Especially for the separation part 14A, because of easiness of capturing blood cells, as shown in
A volume of the fluid storage 24 is preferably in the range from 5 to 5000 mm3. By setting the volume as described above, each of phenomena which take place in the microchannel can be easily controlled. Horizontal sizes of the substrate 12 and the cover plate 22 are not especially restricted, and may be a size suitable for carrying, for example, 80×50 mm, considering easy usage on site of the blood plasma collection tool 10. Also, thicknesses of the substrate 12 and the cover plate 22 are not especially restricted, and may be, for example, approximately 5 mm, respectively, considering strength, economy, and the like.
Material of the substrate 12 is not especially restricted, but because a manufacturing method described below is made easy, resin material, more specifically, polydimethyl sulfoxide (PDMS), polymethyl methacrylate (PMMA), polyvinyl chloride (PVC), ultraviolet curing resin, polycarbonate (PC) etc. may be preferably used.
Material of the cover plate 22 is not especially restricted, but because of visibility for recognizing phenomena in the flow channel, it may be preferably transparent. As such material, various resin boards, more specifically, polydimethyl sulfoxide (PDMS), polymethyl methacrylate (PMMA), polyvinyl chloride (PVC), ultraviolet curing resin, polycarbonate (PC) etc., various resin films, more specifically, polyethylene terephthalate (PET), polyethylene naphthalate (PEN), triacetyl cellulose (TAC) etc., and various glass (soda-lime glass, borosilicate glass etc.) may be used.
It is preferable that the surface of the substrate 12 (the surface on which the long groove is formed) and the bottom surface of the cover plate 22 (the surface which adheres to the substrate 12) keep sufficient flatness for prevention of fluid leakage.
In order to manufacture the substrate 12 having the fine flow channel described above, the fine processing technology may be suitably used. As the fine processing technology, there are, for example, the following technologies.
(1) LIGA technology having a combination of X-ray lithography and electroplating
(2) High-aspect-ratio photolithography using EPON SU8
(3) Mechanical micro-cutting (micro drilling in which a drill having a drill diameter of a micro order is rotated at a high speed)
(4) High-aspect-ratio processing of silicon by Deep RIE
(5) Hot Emboss processing
(6) Laser beam lithography
(7) Laser beam machining
(8) Ion beam processing
Next, a method for firmly attaching the cover plate 22 to the substrate 12 will be described.
First, the substrate 12 and the cover plate 22 are cleaned and subsequently dried. Next, laminating the substrate 12 and the cover plate 22, and they are firmly attached to each other. As for this method for firmly attaching, when material of the substrate 12 and the cover plate 22 is thermoplastic resin, while a laminated body of the substrate 12 and the cover plate 22 is heated to a temperature equal to or higher than their glass transition point Tg, they can be pressurized to be firmly attached to each other. As another method, they can be attached using various adhesives. Further, the operation is preferably conducted in a clean bench or a clean room with cleanliness class being not greater than 100 in view of quality of the blood plasma collection tool 10.
A preferable method for supplying blood into the fluid storage 24 is a method in which blood is supplied by directly touching the fluid storage 24 with a finger tip having blood spilled. Further, there may be a method in which, covering the fluid storage 24 with tape, air expansion caused by pushing down to bend the tape with a finger or heating is used to send fluid. Moreover, there may be also a method for sending fluid using decompression inside the collection part 16A caused by cooling the collection part 16A with ice etc.
Next, procedures for a blood plasma collection method using the blood plasma collection tool 10 according to the present invention will be described with reference to
As shown in
Next, as shown in
Further, as shown in
As shown in
The collected blood plasma 41 is collected externally by a syringe not shown etc. through the collection port 28, and subsequently processed by various analysis and/or inspection equipment.
As described above, using the phenomenon of spontaneous sedimentation of blood cells in blood due to an effect of gravitational force which takes place in the very narrow microspace having the depth in the direction of gravitational force of not greater than 1 mm, blood plasma can be collected by accurately, readily separating blood plasma and blood cells in the small amount of blood from each other in a short time. Further, the separation of blood plasma and blood cells can be conducted by the small tool having a simple structure.
As mentioned above, the preferred embodiments of the blood plasma collection method and tool according to the present invention have been described, but the present invention is not limited to the embodiments described above, and various aspects may be made thereto.
For example, in this embodiment, the width W of the overflow channel 18A in the horizontal direction is made smaller than those of the separation part 14A and the collection part 16A, but not limited to this, the width may be made equal to that of the separation part 14A, and also may be reduced gradually from the upstream side (on the side of the separation part 14A) toward the downstream side (on the side of the collection part 16A). The diameter of the flow channel 20A is not especially limited to the aspect of
Further, in this embodiment, the blood plasma collection tool for separating blood cells from blood to collect blood plasma has been described, but not limited to this, and it may be used as analysis and/or inspection instrument. For example, a diagnostic reagent is applied on an inner wall face of the collection part 16A (the bottom surface etc.) or put into there, and also blood plasma can be introduced into the collection part 16A to be analyzed or inspected on site. In this case, a substrate 12 and a cover plate 22 constituting a blood plasma collection tool 10 are formed of transparent material. Further, in order to observe more easily a phenomenon, a magnifying glass function (lens function) may be provided on the cover plate 22 corresponding to a portion of the collection part 16A. Moreover, when a blood plasma collection tool is used disposably, the collection port 28 may not be necessarily provided.
Further, in this embodiment, the cross section shape of the separation part 14A taken along the line A-A is not limited to the shape of
Further, a position of the inlet 42 of the separation part 14A is not limited to the aspect of
Further, one or more filters or dams may be provided in the overflow channel 18A for communicating the separation part 14A with the collection part 16A.
Further, in this embodiment, an example that only one separation part 14A is provided has been shown, but not limited to this, as shown in
An experiment on collection of blood plasma was conducted using the blood plasma collection tool 10 shown in
Among cell components in blood, most of them are red blood cells (in blood of 1 μL, the number of red blood cells is approximately five million, the number of leukocytes is in the range from 5000 to 10000, and the number of blood platelets is two hundred fifty thousand). Therefore, the separation part 14A was designed, selecting red blood cells as target blood cells to be separated out, and considering the sedimentation time of red blood cells.
Supposing that a diameter of blood cells is 5.7 μm, approximating by a sphere, the spontaneous sedimentation rate vb of red blood cells was 1.6 μm/sec obtained from the expression (1). For fluid characteristics of blood, ρ1=1.09 g/cm3, ρ2=1.0 g/cm3, and η=0.01 poise were used, and the calculation was made with each of these characteristic values being converted to be expressed in suitable units described above.
Blood of 5 μL all was dropped into the fluid storage 24 (diameter: 3 mm, depth: 2 mm) formed in the blood plasma collection tool 10. The fluid storage 24 was covered by applying a thin tape 36 on it and ice was placed on the cover plate 22 on the side of blood plasma collection. Due to this ice, a gas in the collection part 16A was cooled to contract in volume, supplying the blood to the flow channel 20A, the separation part 14A, the overflow channel 18A and the collection part 16A.
At this time, the maximum supply flow rate of blood was 2.4 μL/min. Further, the initial rate vo in the horizontal direction in the separation part 14A was 12 mm/min, and a retention time of blood in the separation part 14A was 2 min.
Reynolds number Re in the separation part 14A was 0.07, which was obtained as the result of calculation using a circle equivalent diameter of the separation part 14A D=4×1×0.2/2×(1+0.2)=0.33 mm and fluid characteristics of blood plasma having a low viscosity (ρ=1.0 g/cm3 and η=0.01 poise), and it was confirmed that a laminar flow was formed. Operation of the blood plasma collection tool 10 ended when the blood plasma reached the collection part 16A.
As the result, the blood cells dropped to 200 μm at a maximum, and could be collected in the separation part 14A. Further, the blood plasma collected in the collection part 16A was visually observed by a microscope to measure a contamination rate of red blood cells, and as the result, the contamination rate of red blood cells was 0%.
Next, an experiment was made similarly to the example 1, except that the supply flow rate of blood was increased to 5 μL/min and the height h of the overflow channel 18A was changed to 105 μm.
At this time, when the initial rate vo in the separation part 14A was 25 mm/min and the retention time of blood in the separation part 14A was t=L/vo=1 min, then the sedimentation distance Hb of blood cells was 96 μm. Further, Reynolds number Re in the separation part 14A was 0.14 and the laminar flow was formed. Operation of the blood plasma collection tool 10 ended when the blood plasma reached the collection part 16A.
As the result, blood cells could be separated out in the separation part 14A and blood plasma could be collected in the collection part 16A. Further, the blood plasma collected in the collection part 16A was visually observed by a microscope to measure the contamination rate of red blood cells, and as the result, the contamination rate of red blood cells was 0%.
Next, an experiment was made similarly to the example 2, except that the height h of the overflow channel 18A was changed to 20 μm.
As the result, in the blood plasma collection tool 10, blood cells entered the collection part 16A and then blood plasma by itself could not be collected. At this time, when the initial rate vo in the separation part 14A was 25 mm/min and the retention time of blood in the separation part 14A was t=L/vo=1 min, then the sedimentation distance Hb of blood cells was 96 μm. Therefore, blood cells which reached the side wall face 46b of the separation part 14A on the downstream side was situated at a position of 104 μm from the bottom of the separation part 14A, and on the contrary, the height h of the overflow channel 18A was 20 μm, so that it was thought that the blood cells flowed into the collection part 16A through the overflow channel 18A.
As mentioned above, it was found that, using the blood plasma collection method and tool according to the present invention, blood plasma could be separated from blood in a short time.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2006-263485 | Sep 2006 | JP | national |
| Number | Name | Date | Kind |
|---|---|---|---|
| 7223364 | Johnston et al. | May 2007 | B1 |
| 20030175167 | Takanori et al. | Sep 2003 | A1 |
| Number | Date | Country |
|---|---|---|
| 1 346 773 | Sep 2003 | EP |
| 2000-171461 | Jun 2000 | JP |
| 2003-83958 | Mar 2003 | JP |
| 2003-270239 | Sep 2003 | JP |
| 2005-292092 | Oct 2005 | JP |
| 2006-52950 | Feb 2006 | JP |
| WO 03008931 | Jan 2003 | WO |
| 2005089082 | Sep 2005 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20080073297 A1 | Mar 2008 | US |
