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.
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 |