JP2003135435A - Blood specimen collecting implement, method of preparing solution for quantification using the same, appliance for preparing solution for quantification and method of using the same and quantification method using solution for quantification - Google Patents

Abstract

PROBLEM TO BE SOLVED: To facilitate blood specimen collection and to facilitate the preparation for a solution for quantification. SOLUTION: This implement has an implement body 1 which is internally formed with a blood housing chamber 2, a puncture body 5 which is freely advanceably and retreatably disposed in this implement body 1, an advancing and retreating movement mechanism 6 which advanceably and retreatably moves this puncture body 5 between an initial position or retreat position and a puncture position, an engaging and disengaging mechanism 7 which freely engageably and disengageably locks the puncture body 5 to the initial position, energy accumulating means 8 which accumulates the driving energy necessary for the puncture body 5 in operating to advance from the initial position to the puncture position and transmits the energy to the advancing and retreating movement mechanism 6 and a pressure reduction mechanism 9 which reduces the pressure in the blood housing chamber 2 to a negative pressure state cooperating with the advancing and retreating movement mechanism 6 in the process that the puncture body 5 operates to retreat from the puncture position to the retreat position. Also, the implement is furnished with an implement body 11 which is internally housed with the first dilute solution 12 and is formed with a dilute blood solution housing chamber 13 of a hermetic and reduced pressure state and a puncture body 15 which is communicated and connected to this dilute blood solution housing chamber 13.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a blood collecting tool used for analyzing and testing components in blood of animals and humans, and in particular, a blood collecting tool effective in facilitating preparation of a quantitative solution. Furthermore, a method for preparing a quantification solution consisting of a diluted plasma solution and / or a quantification solution consisting of a diluted cell fraction solution, a quantification solution preparation instrument and a method of using the same, and this were prepared using this blood collection tool. The present invention relates to a method for quantifying a substance to be measured in plasma and / or a substance to be measured in a cell fraction (particularly, a ratio of hemoglobin A _1C to hemoglobin in a cell fraction) using a quantification solution.

[0002]

2. Description of the Related Art In hospitals, public health centers, test centers, etc., blood is generally collected by a syringe or a vacuum blood collection tube, but these are collected by specialists such as doctors, nurses, and clinical laboratory technicians. The way is done. This type of blood collection method requires the purpose of going out to these facilities, and it is troublesome in that it requires labor, so the fact is that the tests are not frequently adopted for personal health management. In recent years, people with great interest in self-management and health, such as diabetics, often use self-examination for self-examination, and various methods are used. It has been proposed to injure the skin of the skin and collect blood that oozes, or to collect blood from a place with few pain points such as an arm with a handy device.

[0003]

However, in the method of using a syringe and the method of collecting blood that bleeds, it is necessary to apply force by applying a needle or a blade to one's own skin, so that these blood samples are collected. As for the method, the blades and needles were exposed, and applying it directly to the skin of the body was a great mental pain. In particular, since the finger has many pain points, it hurts when it is punctured, which is also a mental pain. In addition, the method of using a handy device (for example, Softat manufactured by Dynabot Co., Ltd. or Atrust manufactured by Teijin Co., Ltd.) is such that blood is collected from a place where there are few pain points such as the arm, and Since the puncture part to the is hidden, mental pain is reduced, but the device itself is expensive.

Further, Japanese Unexamined Patent Publication No. 9-182736 discloses a tool for naturally puncturing a needle with the biasing force of a spring. However, since the device does not have a blood collecting function, a device for collecting blood is required separately.

Further, conventionally, centrifugation has been used as a means for separating blood collected by a blood collecting tool into plasma and cell fractions, and a method using a filter is also known. However, in the method of passing the collected blood as it is through the filter, it takes a long time to pass through the filter due to the physical properties such as blood viscosity, and the obtained plasma may be unstable depending on the components, and it may take time before measurement. If there is a problem, some items were unreliable as inspection values.

The present invention has been made in order to solve the above technical problems, and eliminates mental distress and can easily and surely collect a certain amount of blood (should be solved). The first technical problem is to provide a blood collection tool. Further, the present invention provides a blood collecting tool that can generate a diluted blood solution at the same time as blood sampling and can easily prepare a quantitative solution (second technical problem to be solved). Here, the first technical problem is common to the second technical problem that the quantitative solution can be easily prepared because blood can be easily collected. Furthermore, the present invention also provides a method for preparing a quantitative solution using the blood collecting tool, a preparation instrument and a method for using the same, and a quantitative method using the quantitative solution.

[0007]

That is, the invention for solving the first technical problem is, as shown in FIG. 1 (a), that a blood storage chamber 2 is formed inside and the blood storage chamber is formed. A tool body 1 having a blood suction portion 3 communicating with the chamber 2 for keeping a tight seal with the blood sampling portion 4, and a puncture body movably provided to the tool body 1 to puncture the blood sampling portion 4. 5
An advancing / retreating mechanism 6 that moves the puncture body 5 between an initial position or a retracted position and the puncture position so that the puncture body 5 can advance and retract, and an engagement / disengagement mechanism 7 that engages and disengages the puncture body 5 in the initial position. Energy accumulating means 8 for accumulating drive energy necessary for the puncture body 5 to advance at least from the initial position to the puncture position and transmitting it to the advancing / retreating mechanism 6, and the puncture body 5 retracts from the puncture position to the retracted position. In the process, a decompression mechanism 9 that works in conjunction with the advancing / retreating movement mechanism 6 to decompress the blood storage chamber 2 to a negative pressure state.
A blood sampling tool comprising: (Claim 1). According to the blood collection tool of the present case, a predetermined amount of blood can be collected by the method of automatically puncturing the puncture body 5. In short, since it is automatic puncture, it is not necessary to puncture the skin or the like by itself, and there is little mental distress. Further, since blood can be sucked and stored in the blood storage chamber 2, a certain amount of blood can be secured.

In such technical means, it is necessary that the tool body 1 has at least the blood storage chamber 2 and the blood suction portion 3. Here, the blood storage chamber 2 is
Any room may be used as long as it stores the collected blood, and it does not matter whether or not a diluent storage chamber 10 described later is provided. In addition, the blood suction unit 3
May be anything that communicates with the blood storage chamber 2 and keeps a tight seal with the blood sampling portion 4, such as a suction cup. This is necessary for sucking and storing blood in the blood storage chamber 2.

In particular, as shown by a virtual line in FIG.
It is preferable that the blood containing chamber 2 be provided with a diluent containing chamber 10 containing the first diluent in a fluid-tight manner (claim 2). According to this aspect, it is preferable to generate a diluted blood solution at the same time as blood sampling. The liquid must contain a known concentration of indicator substance. The positional relationship and the functional relationship between the two storage chambers 2 and 10 (the structure in which the blood is gradually mixed with the diluent when blood is collected in the blood storage chamber 2) may be appropriately selected. It is necessary to keep the connecting portion of both liquid-tight so that the diluted liquid does not leak from the liquid storage chamber 10.

Further, the puncture body 5 includes a wide range of needles and blades as long as it can puncture the blood sampling portion 4. On the other hand, as the advancing / retreating movement mechanism 6, as long as the puncture body 5 is advanced / retreated within a predetermined range, a link mechanism may be selected as appropriate. At this time,
It is preferable that the advancing / retreating mechanism 6 is provided with an operating portion that can be manually operated (claim 5). This is the energy storage means 8
In addition to the driving of the puncture body 5 by the stored energy due to, the return operation and the re-extrusion operation of the puncture body 5 can be performed manually, and the usability of the blood collecting tool can be improved accordingly. Further, the initial position and the retracted position of the puncture body 5 may be the same position or may be set at different positions. Further, as the engagement / disengagement mechanism 7, it is sufficient that the puncture body 5 can be locked at the initial position and the locked state of the puncture body 5 can be released in a state where the driving energy by the energy storage means 8 is accumulated.

The energy accumulating means 8 may be any one utilizing physical energy, such as one utilizing deformation by an elastic body such as a spring or rubber, one utilizing a pressure difference of gas, or electromagnetic force. Widely includes those using. Regarding the amount of stored energy, it is sufficient that the puncture body 5 can advance to at least the initial position to the puncture position, which enables automatic puncture by the puncture body 5. Here, in the mode in which the energy accumulating means 8 does not give drive energy for the returning operation of the puncture body 5 to the retracted position, the return operation of the puncture body 5 to the retracted position may be performed manually, for example. Including.

Particularly, as a preferable mode of the energy accumulating means 8, the puncturing body 5 accumulates the driving energy necessary for the advancing operation from the initial position to the puncturing position and the retracting operation from the puncturing position to the retracted position. (Claim 3). According to this aspect, automatic puncture and automatic blood collection are possible. Specifically, since the puncture body 5 automatically retracts after performing the puncture operation, the puncture operation by the puncture body 5 is instantaneously performed. On the other hand, since the decompression mechanism 9 performs the decompression operation with the automatic retraction operation of the puncture body 5, the blood suction operation to the blood storage chamber 2 is automatically performed.

Further, the decompression mechanism 9 is a requirement for sucking and storing blood in the blood storage chamber 2, and a typical example is a mode having a piston portion. The decompression mechanism 9 needs to be interlocked with the advancing / retreating movement mechanism 6, whereby the decompression mechanism 9 can be reliably operated when the puncture body 5 returns from the puncture position to the retracted position. Here, as a typical mode of the decompression mechanism 9 including the piston portion, the decompression mechanism 9 is provided with a piston portion that moves back and forth in the blood storage chamber 2 in association with the forward and backward movement of the puncture body 5 by the forward and backward movement mechanism 6. The blood accommodating chamber 2 is decompressed in conjunction with the retreat operation of the puncture body 5 by the retraction operation of the puncture body 5 and the re-advancement operation of the puncture body 5 from the retracted position to the puncture position by the advancing / retreating mechanism 6 is performed.
The thing which pushes out the blood inside is mentioned (Claim 4). According to this aspect, it is possible to easily collect blood in the blood storage chamber 2 and to easily remove the collected blood from the blood storage chamber 2. In general, with regard to the extraction of the collected blood, it may be transferred to another container, but in this embodiment, the decompression mechanism 9 is used when the collected blood is taken out, and the piston portion of the blood storage chamber 2 is used. The sampled blood is taken out by pushing out the sampled blood.

Further, in the invention for solving the second technical problem, as shown in FIG. 1 (b), the first diluting liquid 12 is contained inside.
And a tool main body 11 in which a dilute blood solution storage chamber 13 in a sealed and depressurized state is formed, and the tool main body 11
Connected to the diluted blood solution storage chamber 13 of the
And a puncture body 15 for guiding blood to the diluted blood solution storage chamber 13 (Claim 6). According to this aspect, a diluted blood solution can be produced at the same time as blood collection.

In such technical means, this embodiment broadly includes a blood collecting device having a diluted blood solution storage chamber 13. Here, the diluted blood solution storage chamber 13 is a chamber in which the diluted blood solution is stored. For example, in the embodiment shown in FIG. 1A, the blood storage chamber 2 and the diluted liquid storage chamber 10 are combined. To do. The depressurized state of the diluted blood solution storage chamber 13 may be formed in advance or may be formed afterwards. At this time, as a typical example of the mode in which the depressurized state is formed afterwards, the tool body 11 may be provided with a volume varying mechanism 14 for varying the volume of the diluted blood solution storage chamber 13 in a sealed state ( Claim 7). Further, the puncture body 5 may be any one that punctures at least the blood sampling portion 4, and the puncture body 5 other than the one having a blood sampling path such as an injection needle (for example, the puncture body 5 shown in FIG. A method of collecting blood without intervention) is also included.

The present invention also relates to the above-mentioned blood collecting device (FIG. 1).
A method of using (a) and (b)) to prepare a quantification solution composed of a diluted plasma solution or a quantification solution composed of a diluted cell fraction solution is also targeted. First, a method for preparing a quantitative solution using the blood collecting tool shown in FIG. In this case, the present invention is, as shown in FIG. 2 (a), a method for preparing a quantitative solution, which is a diluted plasma solution for quantitatively determining a substance to be measured in plasma, by using the collected blood. hand,
(1) Using the blood collecting tool A shown in FIG. 1 (a), blood is collected in the blood chamber 2 and the collected blood is treated with the first diluent 12 containing the indicator substance of known concentration. Step (2) of diluting the diluted blood solution 16 with the separating means B
To separate the plasma and cell fractions to prepare a diluted plasma solution 17 (claim 8).

In this embodiment, the blood collecting tool A has only to include the blood storage chamber 2 in the tool main body 1, and the presence or absence of the diluting liquid storage chamber 10 does not matter. Therefore, the diluting step with the first diluting liquid is performed by the blood collecting tool A (blood containing chamber 2 + diluting liquid containing chamber 1).
It may be carried out in (0) or may be carried out separately using a quantitative solution preparing device. The first diluting liquid mentioned here needs to contain a known concentration of the indicator substance.

Further, according to the present invention, as shown in FIG. 2 (a), a quantification solution comprising a diluted cell fraction solution for quantifying the substance to be measured in the cell fraction using the collected blood. (1) Using the blood collecting tool A shown in FIG. 1 (a), blood is collected in the blood storage chamber 2, and the collected blood is diluted with the first diluent 12. And (2) separating the diluted blood solution 16 into plasma and a cell fraction by the separating means B, and (3) diluting the separated cell fraction with the second diluent 18. And a step of preparing the diluted cell fraction solution 19 (claim 9). When quantifying the substance to be measured in the cell fraction or when measuring the concentration of the substance to be measured, it is necessary to calculate the dilution ratio of the diluted cell fraction solution 19 of the cell fraction. The liquid 18 contains a known concentration of the indicator substance. In addition, the present invention, as shown in FIG. 2 (a), comprises a diluted cell fraction solution for quantifying the ratio of hemoglobin A _1C to hemoglobin in the cell fraction using the collected blood. A method for preparing a solution, comprising: (1) using the blood collecting tool A shown in FIG. 1 (a) to collect blood in the blood storage chamber 2, and collect the collected blood with the first diluent 12. A step of diluting, (2) a step of separating the diluted blood solution 16 into plasma and a cell fraction by a separating means B, and (3) a step of diluting the separated cell fraction with a second diluent 18. And a step of preparing a diluted cell fraction solution 19 (claim 10).

In such an embodiment, the blood collecting tool A is
It suffices that the tool main body 1 includes the blood storage chamber 2, and the presence or absence of the diluent storage chamber 10 does not matter. Therefore, the first diluent 1
The diluting step by 2 may be performed in the blood collecting tool A (blood storage chamber 2 + diluting liquid storage chamber 10), or may be performed separately by a quantitative solution preparation instrument. In addition, usually diluted blood solution 16
When the diluted plasma solution 17 is passed through the separation means B, the diluted plasma solution 17 is also prepared, but it does not matter here whether or not the diluted plasma solution 17 is prepared. However, when preparing a quantification solution consisting of the diluted plasma solution 17, the first diluent 12
It is necessary that a known concentration of indicator substance is contained.

Next, a method for preparing a quantitative solution using the blood collecting tool shown in FIG. 1 (b) will be described. In this case, the present invention is, as shown in FIG. 2 (a), a method for preparing a quantitative solution consisting of a diluted plasma solution for quantitatively quantifying a substance to be measured in plasma using the collected blood. (1)
A step of using the blood collecting tool A shown in FIG. 1 (b) to generate the diluted blood solution 16 in the diluted blood solution storage chamber 13,
(2) separating the diluted blood solution 16 into plasma and cell fractions by a separating means B to prepare a diluted plasma solution 17 (claim 1).
1). Further, according to the present invention, as shown in FIG. 2 (a), the collected blood is used to prepare a quantification solution comprising a diluted cell fraction solution for quantifying the substance to be measured in the cell fraction. In the method, (1) blood is collected in the diluted blood solution storage chamber 13 using the blood collecting tool A shown in FIG. 2B, and the collected blood is diluted with the first diluent 12. Step, (2) a step of separating the diluted blood solution 16 into plasma and a cell fraction by a separating means B, and (3) diluting the separated cell fraction with a second diluent 18 and diluting it. And a step of preparing the cell fraction solution 19 (Claim 12). When quantifying the substance to be measured in the cell fraction or when measuring the concentration of the substance to be measured, it is necessary to calculate the dilution ratio of the diluted cell fraction solution 19 of the cell fraction. The liquid 18 contains a known concentration of the indicator substance. Here, the second diluent 18
May be appropriately selected as long as it dilutes the cell fraction, but is preferably a solution containing a substance that stabilizes the substance to be measured in the cell fraction.

In addition, as shown in FIG. 2 (a), the present invention uses a sample of blood to prepare a diluted cell fraction solution for quantifying the ratio of hemoglobin A _1C to hemoglobin in the cell fraction. And (2) a step of producing a diluted blood solution 16 in the diluted blood solution storage chamber 13 by using the blood sampling tool A shown in FIG. 1 (b), A step of separating the diluted blood solution 16 into plasma and a cell fraction by a separating means B; (3) diluting the separated cell fraction with a second diluent 18 to obtain a diluted cell fraction solution 19. And a step of preparing the same (Claim 13). Here, the second diluent 18
May be appropriately selected as long as it dilutes the cell fraction, but is preferably a solution containing a hemoglobin stabilizer.

Further, the present invention is also directed to a quantitative solution preparing device embodying the quantitative solution preparing method described above.
First, a quantitative solution preparing device using the blood collecting tool shown in FIG. 1 (a) will be described. In this case, the present invention is shown in FIG.
As shown in (b), the blood collecting tool A shown in FIG.
Separation means B for separating plasma and cell fraction from the diluted blood solution after producing a diluted blood solution from the blood collected by the blood collecting tool A, and diluted plasma separated and prepared by the separation means B The container is provided with a quantitative solution container C for accommodating a quantitative solution composed of a solution (claim 14).

Of the blood collecting tools shown in FIG. 1 (b),
A quantification solution preparation instrument using a blood collection tool equipped with the variable volume mechanism 14 will be described. In this case, according to the present invention, as shown in FIG. 2 (b), the blood collecting device A with the variable volume mechanism 14 shown in FIG. And a cell fraction, and a quantitative solution storage container C for storing a quantitative solution consisting of the diluted plasma solution separated and prepared by the separating means B. (Claim 1
5).

Here, as a typical mode of the above-mentioned quantitative solution preparing device, the separating means B is provided at the inlet portion of the quantitative solution container C and holds a cell fraction and allows plasma to pass therethrough. And a communication member that is provided on the inlet side of the filter and is connected to the diluted blood solution storage chamber 13 of the blood collection tool A, and reduces the volume of the diluted blood solution storage chamber 13 by the volume variable mechanism 14. Then, the diluted blood solution in the diluted blood solution storage chamber 13 is separated by the separating means B.
To prepare a quantitative solution containing a diluted plasma solution in the quantitative solution container C (claim 1).
6). Regarding the cell fraction retained on the filter,
By diluting with the second diluent, a quantitative solution consisting of a diluted cell fraction solution can be prepared.

As a method of using this quantitative solution preparing device, a blood collecting tool A with a volume variable mechanism 14 shown in FIG. 1 (b) is used to generate a diluted blood solution in a diluted blood solution storage chamber 13. After that, after connecting the communicating member of the separating means B to the diluted blood solution storage chamber 13 of the blood collection tool A, the volume of the diluted blood solution storage chamber 13 is reduced by the volume variable mechanism 14 of the blood collection tool A to obtain the diluted blood. The diluted blood solution in the solution storage chamber 13 may be passed through the separating means B to prepare a quantitative solution composed of a diluted plasma solution in the quantitative solution storage container C (claim 17).

Further, among the blood collecting tools shown in FIG. 1 (b),
A quantification solution preparation instrument using a blood collection tool in a mode in which the volume of the diluted blood solution storage chamber 13 is constant (a mode in which the first diluent is stored in the vacuum blood collection tube) will be described. in this case,
As shown in FIG. 2 (b), the present invention has a tool main body 11 in which a first diluting liquid is contained and a diluting blood solution accommodating chamber 13 in a sealed and depressurized state is formed in advance.
The blood collecting tool A shown in (b) and the blood collecting tool A,
Separation means B for separating plasma and cell fractions from the prepared diluted blood solution, and a quantification solution storage chamber that is hermetically sealed and at least in a reduced pressure state than the diluted blood solution storage chamber 13 of the blood collection tool A are provided. A container provided with a quantitative solution container C for accommodating a quantitative solution composed of the diluted plasma solution separated and prepared by the means B can be mentioned (claim 18).

In this embodiment, the quantitative solution storage container C is required to be hermetically sealed and in a reduced pressure state as compared with the diluted blood solution storage chamber 13 of the blood collecting tool A, whereby the diluted blood solution is separated by the separating means (filter) B. It moves to the solution container C for quantitative determination via the pressure difference.

Here, as a typical mode of the above-mentioned quantitative solution preparing device, the separating means B is a filter which holds a cell fraction and allows plasma to pass through, and the dilution means which is provided on the inlet side of this filter. An inlet-side communication member that is communicatively connected to the blood solution storage chamber, and an outlet-side communication member that is provided on the outlet side of the filter and that is communicatively connected to the quantification solution storage chamber are provided, and the diluted blood solution storage of the blood collection tool A is provided. An example is one in which the diluted blood solution in the chamber 13 is passed through the separating means B to prepare a quantitative solution containing a diluted plasma solution in the quantitative solution container C (claim 19). The cell fraction retained on the filter can be diluted with the second diluent to prepare a quantification solution consisting of the diluted cell fraction solution.

Further, as a method of using this quantitative solution preparing device, there is provided a tool body in which the first diluting liquid is housed and a diluting blood solution accommodating chamber in a sealed and depressurized state is formed in advance. The blood collecting tool A shown in FIG. 1 (b) is used to generate a diluted blood solution in the diluted blood solution storage chamber 13, and then the diluted blood solution storage chamber 13 of the blood collecting tool A is connected to the inlet side communication member of the separating means B. , And then the outlet-side communication member of the separating means B is connected to the quantification solution storage chamber of the quantification solution storage container C by the pressure difference between the quantification solution storage chamber and the diluted blood solution storage chamber 13. The diluted blood solution in the diluted blood solution storage chamber 13 of the blood collection tool A may be passed through the separating means B to prepare a quantitative solution composed of a diluted plasma solution in the quantitative solution storage container C (claim) Item 20).

The present invention is also directed to a quantification method using the quantification solution prepared by these quantification solution preparation methods. First, to give a typical embodiment of the method for quantifying a component to be measured in plasma, the present invention is a method for quantifying a substance to be measured in plasma using blood as shown in FIG. 3 (a). , (1) a step of preparing a quantification solution consisting of a diluted plasma solution by the quantification solution preparation method described above, (2) the concentration of the indicator substance in the quantification solution, and the instruction in the first diluent From the concentration of the substance, a step of calculating the dilution ratio of plasma in the quantification solution, (3) a step of measuring the concentration of the substance to be measured in the quantification solution, and (4) calculation in (2) And the step of quantifying the concentration of the substance to be measured in the plasma from the dilution ratio and the concentration of the substance to be measured in the quantification solution measured in (3) ( Claim 2
1).

Further, as a typical embodiment of the method for quantifying the components to be measured in the cell fraction, the present invention uses blood as shown in FIG. 3 (b) to measure the components to be measured in the cell fraction. A method for quantifying a substance, comprising the steps of (1) preparing a quantification solution comprising a diluted cell fraction solution by the quantification solution preparation method described above, and (2) the concentration of an indicator substance in the quantification solution. And the second
Calculating the dilution ratio of the cell fraction in the quantification solution from the concentration of the indicator substance in the diluent, and (3) measuring the concentration of the substance to be measured in the quantification solution, (4)
And a step of quantifying the concentration of the substance to be measured in the cell fraction from the dilution ratio calculated in (2) and the concentration of the substance to be measured in the quantification solution measured in (3). It is a feature (claim 22). In addition, representative examples of the method for quantifying the ratio of hemoglobin A _1C , which is a component to be measured, to hemoglobin in the cell fraction are described below.
As shown in FIG. 3 (c), a method of quantifying the ratio of hemoglobin A _1C to hemoglobin in the cell fraction using blood, comprising: And a step of quantifying the ratio of hemoglobin A _1C to hemoglobin in the quantification solution prepared in (2) and (1). (Claim 2
3).

[0032]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described below in detail based on the embodiments shown in the accompanying drawings. ◎ Embodiment 1 FIG. 4 is a cross-sectional explanatory view of Embodiment 1 of a blood collecting device to which the present invention is applied, and FIG. 5 is a side view thereof. In the present embodiment, blood collection tool 100 has a tool body 110 having blood storage chamber 112, a puncture blade 130 as a puncture body that is provided to be movable back and forth with respect to this tool body 110, and forward and backward movement of puncture blade 130. An advancing / retreating movement mechanism 140,
An energy storage member 160 for supplying drive energy to the advancing / retreating movement mechanism 140, an engagement / disengagement mechanism 170 for engaging / disengaging the advancing / retreating movement mechanism 140 to an initial position, and an advancing / retreating movement mechanism 14.
A decompression mechanism 1 for decompressing the blood storage chamber 112 in cooperation with 0
80 and.

In the present embodiment, the tool body 110
Is, for example, a box-shaped synthetic resin housing 11
1 is provided, and a cylindrical cylinder portion 113 that defines a blood storage chamber 112 is provided on the top side of the housing 111, and a nozzle portion 114 that communicates with the blood storage chamber 112 is provided outside the cylinder portion 113. It is formed by projecting. Here, the blood storage chamber 112 may be appropriately selected according to the amount of blood to be collected (usually a small amount of 1 mL or less), and may have a volume of, for example, about 5 mL or less. A part of the side surface of the housing 111 is formed by a removable cover 115 as shown in FIGS. 5 and 6 (a). Further, a rubber suction cup 116 is provided at the tip of the nozzle 114,
For example, it is adapted to be adsorbed and fixed around the blood sampling portion 190 (see FIG. 11) of the human body.

On the outside of the cylinder portion 113,
As shown in FIGS. 4 and 8, a diluent storage ring 118, which defines a doughnut-shaped and channel-shaped diluent storage chamber 119, is fitted and mounted by a screw or the like, and the cylinder 113 and the diluent are connected. A communication hole 1 is provided between the housing ring 118 and the housing ring 118.
20 (In this example, the shape of the communication hole 120 on at least one side is a long hole shape, and a structure for ensuring a communication state between the both is adopted)
Are provided in an appropriate number (for example, four places), and a seal member 121 such as an O-ring is interposed between the cylinder portion 113 and the diluent storage ring 118. Incidentally, FIG. 6 (a)
Reference numeral 123 is a seal groove in which the seal member 121 is housed. The opening of the diluent storage ring 118 is liquid-tightly provided to the housing 111 by a ring-shaped rubber seal 122. Furthermore, the diluent storage chamber 1
In consideration of the agitating property, the first diluting liquid 19 is pre-stored in 19 in an amount smaller than full. Here, the first diluting liquid 21 will be described in detail when explaining the preparation of the quantitative solution of the second embodiment.

Further, the puncture blade 130 is usually made of metal,
Steel and stainless steel are common. The puncture blade 130 is arranged in the nozzle portion 114 so as to be movable back and forth, and is connected to a moving back and forth mechanism 140. In the present embodiment, the advancing / retreating movement mechanism 140 includes a rotating plate 141 rotatably arranged in the housing 111, and the rotating plate 141.
And a link mechanism 142 that is pin-connected to a position deviated from the center position of the puncture blade 130 and is pin-connected to the puncture blade 130, and the puncture blade 130 is moved forward and backward with the rotation of the rotary plate 141. . Here, the link mechanism 1
As shown in FIG. 7A to FIG. 7D, for example, two link arms 143, 144 are pin-coupled to each other in a form of fitting a boss 145 and an emboss 146, and the tip of one link arm 143 is connected. Puncture blade 130 on the side with pin 14
7 is connected and an elastically deformable joining pin 148 is provided on the tip side of the other link arm 144,
41 to the connecting hole 149 (see FIG. 9A).
The one in which 8 is elastically fitted is used. Also, the rotating plate 1
In the vicinity of 41, the stopper claw 15 that the joint pin 148 of the rotary plate 141 abuts to regulate the rotation range of the rotary plate 141.
6 is provided.

Further, in the present embodiment, the rotary plate 14
As shown in FIG. 9A, the rotary shaft 150 of No. 1 is provided with an impeller-shaped driving force transmission portion 151, and the tip of the rotary shaft 150 is exposed to the outside of the housing 111.
An operation handle 153 is connected to the tip of the rotary shaft 150 so as to be manually operable. Further, the energy storage member 16
As 0, for example, rubber or a spring material is used, and the energy storage member 160 is disposed in engagement with the driving force transmission portion 151 so that the rotational driving force is applied to the rotary plate 141.
Furthermore, a locking protrusion 152 is provided on a part of the periphery of the rotary plate 141.

Further, in this embodiment, the engagement / disengagement mechanism 1
The 70 includes a locking member 171 that is erected on the bottom of the housing 111 and tilts within an elastically deformable range, and a locking release mechanism 174 that tilts the locking member 171. In the present embodiment, the locking member 171 is, as shown in FIGS. 4 and 9B, the locking projection 1 of the rotary plate 141.
A locking protrusion 172 that engages with and disengages from 52 is provided, and an elastic concave portion 173 that serves as a tilt fulcrum for a lateral load is provided at the base end portion thereof. On the other hand, the lock release mechanism 17
4 is a housing 1 as shown in FIGS.
A rod mounting portion 175 is provided in a part of the rod 11, a rod insertion hole 176 is provided in the rod mounting portion 175, and a locking rod 177 is inserted into the rod insertion hole 176 so as to be movable back and forth. An elastically deformable positioning protrusion 178 is provided on a part of the rod mounting portion 175.
The locking rod 177 can be engaged with and disengaged from the positioning hole 179 provided in the locking member 171.
By pushing the locking member 171 toward the side, the locking member 171 is tilted toward the pushing direction of the locking rod 177, and the rotating plate 1
41 and the locking member 171 between the locking projections 152, 172
The locked state is released. In the present embodiment, when the locking projections 152 and 172 are in the locked state between the rotary plate 141 and the locking member 171, the puncture blade 130 is arranged at the initial position. Has become. Further, the locking rod 177 is adapted to return to its original initial position by the elastic restoring force of the locking member 171 when the pushing operation force is released.

Further, in this embodiment, the pressure reducing mechanism 1
As shown in FIGS. 4 and 6B, reference numeral 80 denotes a piston portion 181 arranged in the cylinder portion 113 so as to be movable back and forth.
At the center of the piston portion 181, the link arms 143 and 144 of the link mechanism 142 and the puncture blade 130 are provided.
Through hole 182 through which the piston part 181 is inserted.
Sealing members 183 and 184 made of O-rings are provided in the through-holes 182 of the above. The seal member 183 is wound around the puncture blade 130 side of the link arm 143 of the link mechanism 142 and arranged at the connecting portion between the cylinder portion 113 and the nozzle portion 114.
4 is disposed between a stopper ring 155 (see FIG. 7A) fixed to the link arm 143 of the link arm 143 of the link mechanism 142 and fixed to the link arm 143, and the piston portion 181. . In addition, FIG.
As shown in (b), two seal grooves 185 are provided around the piston portion 181, and the seal groove 185 has an O
A seal member 186 such as a ring is wound around the cylinder portion 1
It is designed to keep the space between 13 and the liquid tight. The material of the housing 111, the piston portion 181 and the like may be appropriately selected in consideration of slipperiness and elasticity of a seal member such as an O-ring. , Phenol resin, polyester, nylon resin, polystyrene, acrylic, polyurethane, synthetic rubber, fluororesin, silicone resin, and the like, which may be selected from commercially available polymers.

Next, a method of using the blood collecting tool according to this embodiment will be described. Now, as shown in FIG. 11, the blood collecting tool 100 is set in a state in which the suction cup 116 is sucked so as to cover the blood sampling portion 190 of the human body. In this state, the engagement / disengagement mechanism 170 holds the locked state, positions the puncture blade 130 at the initial position, and at the same time, stores the energy storage member 16.
0 is held in a state in which the rotary driving force is applied to the rotary plate 141. After this, the locking rod 177 of the engagement / disengagement mechanism 170.
When the pushing operation is performed, the rotating plate 141 and the locking member 171 are
The locking state between the locking projections 152 and 172 between the two is released, and the rotational driving force from the energy storage member 160 is transmitted to the rotary plate 141 via the driving force transmission portion 151, and the rotary plate 141 is moved in the arrow direction. Rotate to.

At this time, in the present embodiment, the rotary plate 14
When 1 rotates about 1/4, the rotary plate 141 and the link mechanism 1 are rotated.
The pin connection part with 42 moves to the most raised position. Then, the puncture blade 130 connected to the link mechanism 142.
Is projected from the nozzle portion 114, and the blood collecting portion 190 of the human body
Is punctured by. In this case, the puncture blade 130 is originally hidden in the nozzle portion 114, and the puncture blade 130 is momentarily released only by performing the unlocking operation of the engagement / disengagement mechanism 170.
Since 0 is punctured, the user who is using the blood collection device 100 is not mentally frightened.

In particular, in the present embodiment, if the energy from the energy accumulating member 160 gives a rotational driving force to the rotary plate 141 until the pin connecting portion of the rotary plate 141 contacts the stopper claw 156. As shown in FIG. 12, the rotary plate 141 is forcibly stopped by the stopper claws 156 after rotating about 3/4 rotation.
When 1 rotates about 1/4 rotation or more, the puncture blade 130 retracts from the puncture position to the retracted position. Therefore, since the puncture blade 130 punctures the blood sampling portion 190 instantaneously, the puncture operation by the puncture blade 130 is less likely to cause a great pain to the user.

Further, as shown in FIG. 12, the puncture blade 130
When the retracting operation is performed, the seal member 183 wound around the link mechanism 142 collides with the link mechanism 142 against the top of the piston portion 181. Then, the piston part 1
81 is the cylinder portion 113 via the seal member 183.
As the piston portion 181 moves, the blood storage chamber 112 sealed by the suction cup 116 is depressurized. In this state, the blood is sucked and stored from the blood sampling portion 190 damaged by the puncture blade 130 to the blood storage chamber 112 side by the negative pressure through the gap between the nozzle portion 114 and the puncture blade 130.

When the piston portion 181 retreats to some extent, the blood storage chamber 112 and the diluent storage chamber 119 are brought into communication with each other through the communication hole 120, and the blood storage chamber 1
Blood M sucked and stored in 12 is sequentially diluted with the first diluent 21. At this stage, the blood collection device 10
A predetermined amount of diluted blood solution is stored in the zero blood storage chamber 112.

After this, when a quantitative solution is prepared using the diluted blood solution collected by the blood collecting tool 100, as shown in FIG. 13, a separate container 30 containing the diluted blood solution is placed. Prepare the blood collection tool 1 so that it faces it.
Place 00. When the operation handle 153 is rotated in a predetermined direction in this state, the puncture blade 130 moves forward through the link mechanism 142, and the seal member 184 abuts on the bottom side of the piston portion 181 through the link mechanism 142. The piston portion 181 moves in the cylinder portion 113 in the pushing direction via the seal member 184. Then, along with the pushing operation of the piston portion 181, the diluted blood solution in the blood storage chamber 112 is pushed out by the piston portion 181, and transferred to the container 30. After that, the work of preparing the quantitative solution is performed using the diluted blood solution in the container 30 (see Embodiment 2).

In the present embodiment, the blood collecting tool 1
As 00, the one provided with a diluent storage chamber 119 connected to the blood storage chamber 112 is used, but the invention is not limited to this, and as shown in FIG. The embodiment in which the storage chamber 119 is not provided may be applied. In this embodiment, undiluted blood is stored in the blood storage chamber 112. After being diluted, it is preferable to prepare the quantitative solution.

[Embodiment 2] In this embodiment, a method for preparing a quantitative solution using the blood collection tool 100 according to Embodiment 1 will be described.
In the present embodiment, the quantitative solution preparing device includes blood collecting tool 100 used in the first embodiment and the quantitative solution preparing tool shown in FIG. In the present embodiment, the quantitative solution preparation tool is a tool for preparing a quantitative solution for quantifying a substance to be measured in plasma or a substance to be measured in a cell fraction, and as shown in FIG. To
It is composed of a first container 30 and a second container 40 which can be fitted to each other. 15A is a front view and a plan view of the first container 30, and FIG. 15B is a front view and a plan view of the second container 40. Here, the first container 30 and the second container 40 are preferably made of plastic, and examples of the material thereof include polyethylene, polypropylene, polycarbonate, vinyl chloride, polyester, nylon, polybutene, polybutadiene, and copolymers thereof. To be

First, the first container 30 will be described.
The first container 30 has, for example, a cylindrical container body 31,
The container body 31 has a diluted blood solution 22 (see FIG. 16).
Alternatively, a diluted blood solution storage portion 32 (corresponding to a portion defining the diluted blood solution storage chamber 33) in which the second diluted liquid 28 (see FIG. 17) is stored is provided, and the diluted blood solution storage portion 32 is provided.
On the inner wall of the container, in other words, on the inlet side of the wall surface of the diluted blood solution storage chamber 33, a female screw portion 34 is formed, while the container body 3
A fixing stud 35 protruding downward is formed on the bottom of the fixing member 1, and the fixing stud 35 is fixedly installed in the fixing holder by engaging and fixing the fixing stud 35 in a screw hole portion of a fixing holder (not shown). . A flange portion 36 is provided at the inlet of the container body 31 so that the step portion between the quantitative solution storage portion 42 and the fitting portion 44 of the second container 40, which will be described later, abuts.

On the other hand, the second container 40 has, for example, a cylindrical container body 41, and the container body 41 contains a quantitative solution (diluted plasma solution or diluted cell fraction solution). A portion 42 (corresponding to a portion that defines the quantification solution storage chamber 43) is provided, and a portion adjacent to the quantification solution storage portion 42 projects toward the first container 30 side and is provided in the first container 30. A fitting portion 44 that fits into the diluted blood solution storage portion 32 is provided, and a communication portion 45 that communicates from the tip toward the quantitative solution storage portion 42 is provided in the fitting portion 44.
Is formed. In the present embodiment, the fixed-quantity solution storage portion 42 includes a storage chamber portion that is hollowed out in a columnar shape, and a storage chamber portion that is hollowed out in a conical shape so as to be adjacent to the lower portion and constrict downward. And a quantitative solution storage chamber 43 composed of The fitting portion 44 has an outer diameter smaller than that of the fixing solution accommodating portion 42, and has a male screw portion 46 screwed onto the female screw portion 34 of the diluted blood solution accommodating portion 32 of the first container 30 on its peripheral wall. It was formed.
Further, the communication portion 45 is connected to the filter holding hole 47 formed from the tip end side of the fitting portion 44 to have a predetermined inner diameter, and the lower end portion of the quantitative solution storage chamber 43 having a smaller diameter than the filter holding hole 47. And a small-diameter connection hole 48 to be formed.

Further, in the present embodiment, the second container 4
A filter 60 is inserted and held in the filter holding hole 47 of No. 0, and the filter 60 is arranged so as to project from the filter holding hole 47 by a predetermined amount m. In the present embodiment, the filter 60 is composed of, for example, a fine fiber aggregate, a porous polymer or a fine particle aggregate, and in an aspect using the fine fiber aggregate or the fine particle aggregate, for example, other than plasma. Cell fraction is captured, porous polymer,
In an embodiment using a fine fiber aggregate in which a porous polymer is mixed or a fine particle aggregate in which a porous polymer is mixed, a cell fraction is retained on the surface, and a diluted plasma solution is retained inside. There is. Incidentally, among the porous polymers,
In particular, examples of materials having excellent high water absorption include polyacrylic acid resins, polyethylene oxide resins, vinyl acetate copolymer resins, acrylic acid graft copolymer resins, and the like. In particular, in the present embodiment, the sum of the length dimension k of the fitting portion 44 and the projection dimension m of the filter 60 from the fitting portion 44 is the sum of the diluted blood solution storage portion 3 of the first container 30.
It is set corresponding to the depth dimension n of 2. Further, an O as a peripheral sealing member is provided around the tip side of the fitting portion 44.
A ring 49 is provided.

Furthermore, in the present embodiment, the second container 4
A lid member 50 is provided on the upper side of the fixed amount solution storage unit 42 of 0 to cover the upper side of the fixed amount solution storage unit 42 in an openable manner. The lid member 50 has a concave portion 51 that fits into the upper protruding portion 42a of the quantitative solution container 42, and the inner surface of the concave portion 51 is screwed to the male screw portion 42b formed on the upper protruding portion 42a. The female screw portion 52 is provided. The lid member 50 has a minute atmosphere opening 53 that communicates with the atmosphere. Furthermore, in the present embodiment, a flange-shaped support piece 54 is provided on the outer periphery of the quantitative solution container 42 of the container body 41. The support piece 54 is for supporting the preparation tool at the installation location of the sample table when the preparation tool of the present case is installed in an automatic analyzer, for example.

The meanings of the terms used in the present embodiment and the present case are clarified as follows. (Plasma and Cell Fraction) Blood is composed of plasma and cell fractions, and the cell fraction contains red blood cells, white blood cells, platelets and the like. In the present invention, plasma represents not only plasma in the original meaning but also serum in the original meaning excluding fibrin aggregated by a blood coagulation reaction. Hereinafter, serum and plasma will be collectively referred to as plasma. Further, in the present invention, when a blood coagulation reaction occurs, the cell fraction also represents a blood clot. Hereinafter, the cell fraction and the blood clot when a blood coagulation reaction has occurred are collectively referred to as a cell fraction.

(Substance to be measured in plasma, substance to be measured in cell fraction) As the substance to be measured in plasma, any of biochemical items and immunity items can be applied. Examples of biochemical items include glucose, 1,5-anhydroglucitol, fucose, urea, ammonia, creatinine, total cholesterol, HDL cholesterol, LDL.
Cholesterol, RLP cholesterol, triglyceride, phospholipid, Na, K, Cl, Ca, total protein, albumin, globulin, bilirubin, bile acid, sialic acid,
Free fatty acids, alanine aminotransferase (AL
T), aspartate aminotransferase (AS
T), creatine phosphokinase (CPK), phosphokinase (PK), amylase, lipase, cholinesterase, γ-glutamyl transpeptidase, L-lactate dehydrogenase (LDH), aldolase, alkaline phosphatase, acid phosphatase and the like. Examples of immunity items include IgG, IgM, Ig
A, IgE, apoprotein AI, apoprotein AII, apoprotein B, apoprotein E, rheumatoid factor, D-dimer, oxidized LDL, glycoalbumin, T3, T4, drugs such as anti-tencan agent, α-fetoprotein (AF
P), carcinoembryonic antigen (CEA), CA19-9, CA-
125, BNP, troponin T, troponin I, human chorionic gonadotropin (hCG), insulin, C-peptide, estrogen, anti-GAD antibody, pepsinogen, HB antigen, anti-HB antibody, HCV antigen, anti-HCV antibody, HTLV-I antigen , Anti-HTLV-I antibody, HIV antibody, tuberculosis antibody, mycoplasma antibody and the like. Examples of the substance to be measured in the cell fraction include hemoglobin A _{1C and the} like.

(First Diluting Solution) The first diluting solution is used for diluting blood, which is used when measuring a substance to be measured in plasma.
It is an aqueous solution for dissolution and an indicator solution for calculating the dilution ratio is contained at a constant concentration. (First Aqueous Medium for Preparing Diluting Solution) The aqueous medium used for preparing the first diluting solution is not particularly limited as long as it stably preserves blood without hemolyzing, for example, Isotonic solutions, physiological saline solutions, buffer solutions and the like can be mentioned. Examples of the isotonic solution include lock solution, Ringer's solution, Tyrode's solution, Earle's solution, Krebs solution, phosphate buffered saline (PBS) and the like. The concentration of sodium chloride in physiological saline is 0.1 to 0.2 mol / L. The buffer used in the buffer solution is not particularly limited as long as it has a buffering ability, and for example, pH 2 to 11, for example, lactate buffer, citrate buffer, acetate buffer, succinate buffer, phthalate buffer, Phosphate buffer, triethanolamine buffer, diethanolamine buffer, lysine buffer, barbitur buffer, tris (hydroxymethyl) aminomethane buffer, imidazole buffer, malate buffer, oxalate buffer, glycine buffer Agent, borate buffer, carbonate buffer, glycine buffer, MES (2-morpholinoethanesulfonic acid) buffer, bis-tris [bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane] buffer, ADA [ N- (2-acetamido) iminodiacetic acid] buffer, PIPES [piperazine-N, N'-
Bis (2-ethanesulfonic acid)] buffer, ACES {2
-[N- (2-acetamido) amino] ethanesulfonic acid} buffer, MOPSO (3-morpholino-2-hydroxypropanesulfonic acid) buffer, BES {2- [N,
N-bis (2-hydroxyethyl) amino] ethanesulfonic acid} buffer, MOPS (3-morpholinopropanesulfonic acid) buffer, TES <2- {N- [tris (hydroxymethyl) methyl] amino} ethanesulfonic acid > Buffer, HEPES [N- (2-hydroxyethyl)-
N '-(2-sulfoethyl) piperazine] buffer, DI
PSO {3- [N, N-bis (2-hydroxyethyl)
Amino] -2-hydroxypropanesulfonic acid} buffer, TAPSO <2-hydroxy-3-{[N-tris (hydroxymethyl) methyl] amino} propanesulfonic acid> buffer, POPSO [piperazine-N, N'- Bis (2-hydroxypropane-3-sulfonic acid)] buffer, HEPPSO [N- (2-hydroxyethyl)-
N '-(2-hydroxy-3-sulfopropyl) piperazine] buffer, EPPS [N- (2-hydroxyethyl) -N'-(3-sulfopropyl) piperazine] buffer, tricine [N-tris (hydroxy) Methyl) methylglycine] buffer, bicine [N, N-bis (2-hydroxyethyl) glycine] buffer, TAPS {3- [N-
Tris (hydroxymethyl) methyl] aminopropanesulfonic acid} buffer, CHES [2- (N-cyclohexylamino) ethanesulfonic acid] buffer, CAPSO [3
Good buffer agents such as-(N-cyclohexylamino) -2-hydroxypropanesulfonic acid] buffer agent and CAPS [3- (N-cyclohexylamino) propanesulfonic acid] buffer agent may be mentioned. The concentration of the buffer solution is not particularly limited, but is preferably 0.1 to 1000 mmol / L.
-500 mmol / L is more preferable.

(Additives in First Diluting Solution) The first diluting solution may optionally contain additives such as a chelating agent, an enzyme stabilizer, an antiseptic, and a coagulation accelerator. . Examples of chelating agents include ethylenediaminetetraacetic acid (ED
TA) and the like, and examples of the enzyme stabilizer include, for example,
N-acetyl cysteine (N as a stabilizer of CPK
Α) as a stabilizer for AC), ALT, AST, CPK
-A ketoglutaric acid etc. are mentioned. Examples of the preservative include sodium azide and the like. Examples of the coagulation accelerator include thrombin and lectin. The origin of the lectin includes, for example, abrin, azuki bean, mokuwanju, siberian beans, sea green algae, red beans, soybeans, sweet pea, lentils, horseshoe crabs, tomatoes, osage oranges, avocados, safflowers, red kidney beans, and American beans. Examples include pokeweed, peas, wing beans, castor beans, potatoes, wheat malt, broad beans, and European mistletoe.

Examples of the extrinsic indicator include dyes,
Examples thereof include a reduction coloring type chromogen, an oxidation coloring type chromogen, a fluorescent substance and a luminescent substance. Examples of the oxidative chromogenic chromogen include a leuco chromogen and an oxidative coupling chromogenic chromogen. Among these, the oxidative coupling coloring type chromogen is preferable.

(Dye) As the dye, for example, Acid Yellow 3, Acid Yellow 23, Acid Yellow 25, Acid Yellow 36, Acid Orange 5, Acid Orange 6, Acid Orange 7, Acid Orange 10, Acid Orange 19, Acid Orange. 5
2, Acid Green 16, Acid Green 25, Acid Violet 43, Acid Blue 3, Acid Blue 9 (Brilliant Blue FCF), Acid Blue 40, Acid Blue 45, Acid Blue 47, Acid Blue 59, Acid Blue 74, Acid Blue 113 , Acid Blue 158, Acid Red 1,
Acid Red 2, Acid Red 14, Acid Red 18, Acid Red 27, Acid Red 37, Acid Red 51, Acid Red 52, Acid Red 87, Acid Red 88, Acid Red 92, Acid Red 94, Acid Red 95, Acid Red 111, Food Red 17, Food Yellow 3, Basic Yellow 1, Basic Yellow 2, Basic Yellow 11, Basic Orange 1, Basic Orange 22, Basic Green 4 (Malachite Green), Basic Violet 3, Basic Violet 4, Basic Violet 10, Basic Blue 1, Basic Blue 3, Basic Blue 9, Basic Blue 24, Basic Red 1, Basic Red 2, Basic Head 5, Basic Red 9, Basic Red 18, and the like.

(Reduction-coloring type chromogen) Examples of the reduction-coloring type chromogen include 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium.
Bromide (MTT), 2- (4-iodophenyl) -3
-(4-Nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium monosodium salt (WST-1), 2- (4-iodophenyl) -3-
(2,4-dinitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium monosodium salt (WST-3) and the like can be mentioned. (Oxidative chromogenic chromogen ~ leuco chromogen) A leuco chromogen is a chromogen that is independently converted into a dye in the coexistence of a hydrogen peroxide and a peroxidative active substance such as peroxidase,
For example, 10-N-carboxymethylcarbamoyl-
3,7-Bis (dimethylamino) -10H-phenothiazine (CCAP), 10-N-methylcarbamoyl-
3,7-Bis (dimethylamino) -10H-phenothiazine (MCDP), N- (carboxymethylaminocarbonyl) -4,4'-bis (dimethylamino) diphenylamine sodium salt (DA-64), 4,4'- Examples thereof include bis (dimethylamino) diphenylamine and bis [3-bis (4-chlorophenyl) methyl-4-dimethylaminophenyl] amine (BCMA).

(Oxidative Chromogen to Oxidative Coupling Chromogen) In the oxidative coupling chromogen, two compounds are oxidized in the presence of hydrogen peroxide and a peroxidative substance such as peroxidase. It is a chromogen that is mechanically coupled to form a dye. Examples of the combination of the two compounds include a combination of a coupler and anilines, and a combination of a coupler and phenols. Examples of the coupler include 4-aminoantipyrine (4-A
A), 3-methyl-2-benzothiazolinone hydrazine and the like. Anilines include N-ethyl-N
-(3-Methylphenyl) -N'succinylethylenediamine (EMSE), N- (3,5-dimethoxyphenyl) -N'succinylethylenediamine sodium salt (DOSE), N-ethyl-N- (2-hydroxy-3)
-Sulfopropyl) -m-toluidine, N-ethyl-N
-Sulfopropylaniline, N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline, N-sulfopropyl-3,5-dimethoxyaniline, N-ethyl-N-
Sulfopropyl-3,5-dimethylaniline, N-ethyl-N-sulfopropyl-m-toluidine, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-
Anisidine, N-ethyl-N- (2-hydroxy-3-
Sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt dihydrate (TOOS), N-ethyl-N
-(2-Hydroxy-3-sulfopropyl) -3,5-
Dimethoxyaniline, N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline sodium salt (HSDA), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5- Dimethylaniline,
N-sulfopropylaniline, N-ethyl-N-sulfopropylanilinepropyl-m-anidin, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -4-
Examples thereof include fluoro-3,5-dimethoxyaniline sodium salt (F-DAOS). Examples of phenols include phenol and 3-hydroxy-2,4,6-triiodobenzoic acid.

(Fluorescent substance and luminescent substance) Examples of the fluorescent substance include 4-hydroxyphenylacetic acid and 3- (4-
Hydroxyphenyl) propionic acid, coumarin and the like can be mentioned. Examples of the luminescent substance include luminol, isoluminol, lucigenin, acridinium ester and the like.

(Second Diluting Solution) As the second diluting solution,
For example, a hypotonic solution such as distilled water or an aqueous solution having a low salt concentration can be used. If necessary, the second diluent contains an indicator substance,
It contains a hemoglobin stabilizer, a surfactant, a lipid hydrolase and the like. An indicator substance is contained when the substance to be measured in the cell fraction is quantified, particularly when the substance to be measured in the cell fraction is measured as a concentration. The above-mentioned is mentioned as an indicator substance. When the ratio of hemoglobin A _1C to hemoglobin in the cell fraction is quantified, it is preferable that the second diluent contains a hemoglobin stabilizer. (Hemoglobin stabilizer) Examples of the hemoglobin stabilizer include sugars, amino acids, protease inhibitors,
Examples thereof include chelating agents, iron protoporphyrin, sodium fluoride, albumins, haptoglobulin and the like.
As the surfactant, for example, a nonionic surfactant,
Examples include cationic surfactants, anionic surfactants, amphoteric surfactants and the like. As a lipid hydrolase,
Examples include phospholipases and lipoprotein lipases.

(Filter) In this embodiment, the filter 60 is used as the separating means. This filter 6
Examples of 0 include a fine fiber aggregate, a porous polymer or a fine particle aggregate. (Fine fiber aggregate) As the fine fiber aggregate, for example,
Examples thereof include glass fiber aggregates, cellulose fiber aggregates, and synthetic fiber aggregates. As the material of the synthetic fiber aggregate,
Examples thereof include polyethylene, polypropylene, polystyrene, polyamide, polysulfone, polyurethane, polyvinyl formal, polyvinyl chloride, and polyesters. If necessary, a hydrophilic fine fiber aggregate may be used. (Porous Polymer and Aggregate of Fine Particles) Examples of the porous polymer include thermosetting polymers (phenolic type, urea type, melamine type, alkyd type, polyester isocyanate type), thermoplastic polymers (polyvinyl formal, polyvinyl chloride). , Polystyrene, polyethylene),
Examples thereof include cellulose derivatives (ester form, ether form, viscose) and the like. As the fine particle aggregate, for example,
Examples thereof include silica fine particle aggregates.

Next, a method of using the quantitative solution preparing tool according to the present embodiment will be described. (Preparation method 1 of quantitative solution consisting of diluted plasma solution)
It is assumed that the embodiment model in which the fine fiber aggregate is inserted and held as the filter 60 in the filter holding hole 47 of the second container 40 is used. In this case, as shown in FIG. 16A, a diluted blood solution (for example, refer to the first embodiment) 22 is stored in the diluted blood solution storage portion 32 of the first container 30,
Then, as shown in FIGS. 16B and 16C, the first container 30 and the second container 40 are fitted to each other, and a fixed amount of the diluted plasma solution 25 is contained in the fixed amount solution storage portion 42 of the second container 40. Prepare a working solution.

In the case of this example, when the fitting portion 44 of the second container 40 is fitted into the diluted blood solution storage portion 32 of the first container 30 and the tip of the filter 60 is immersed in the diluted blood solution 22, While the second container 40 is being pushed into the first container 30, since the atmosphere opening port 53 of the lid member 50 is opened, air escapes from this atmosphere opening port 53, and the diluted blood solution 22 passes through the filter 60. It moves to the side of the quantitative solution container 42. At this time, since the cell fraction S in the diluted blood solution 22 is retained on the surface of the fine fiber assembly which is the filter 60, the plasma in the diluted blood solution 22 is filtered by the filter 6.
After passing through 0, the solution moves to the quantitative solution container 42. Therefore, the quantitative solution containing the diluted plasma solution 25 is prepared in the quantitative solution container 42. Further, as shown in FIG. 15, the small-diameter connection hole 48 of the communication part 45 connects the filter 60 held in the filter holding hole 47 and the fixed quantity solution storage part 42, but the diameter thereof is very small. Because it is thin,
The quantitative solution in the quantitative solution container 42 and the liquid remaining in the filter 60 do not coexist.

(Preparation Method 1 of Quantitative Solution Consisting of Diluted Cell Fraction Solution) Now, an embodiment model in which a fine fiber aggregate is inserted and held as a filter 60 in the filter holding hole 47 of the second container 40 is used. Imagine a case. in this case,
As shown in FIG. 16A, the diluted blood solution 22 is stored in the diluted blood solution storage portion 32 of the first container 30, and then, as shown in FIGS. 16B and 16C, the first container 30 is stored. The second container 40 and the second container 40 are fitted to each other to prepare a quantitative solution composed of the diluted plasma solution 25 in the quantitative solution container 42 of the second container 40. Then, as shown in FIG. Container 3
0 and the fitting state of the second container 40 are released, the quantification solution consisting of the diluted plasma solution 25 is removed from the quantification solution container 42 of the second container 40, while the diluted blood of the first container 30 is removed. A predetermined amount of the second diluent 28 is stored in the solution storage portion 32, and then the first container 30 and the second container 40 are fitted to each other as shown in FIGS. Two
The diluted cell fraction solution 2 is placed in the quantification solution container 42 of the container 40.
A quantitative solution consisting of 7 is prepared.

In such a preparation method, at the stage of preparing the diluted cell fraction solution 27, the quantitative solution container 42 of the second container 40 was washed so that the diluted plasma solution 25 would not affect it. It is preferably used later. Further, although the first container 30 stores the second diluent 28, in order to prevent the influence of the diluted blood solution 22, the first container 30 is also washed with the second diluent 2.
8 or the first container 30
It is preferable to use a separate part. In this preparation method, when the first container 30 and the second container 40 are fitted to each other as shown in FIGS.
8 dilutes the cell fraction S retained on the surface of the filter 60, and moves it to the quantitative solution container 42.

[Embodiment 3] FIG. 18 shows Embodiment 3 of a quantitative solution preparing device to which the present invention is applied. In this figure, the quantitative solution preparing device is a blood sampling device 200 shown in FIG.
It comprises a quantitative solution preparation tool 210 shown in (b). In the present embodiment, blood collection tool 200 has blood collection tube 201.
A diluted blood solution storage chamber 202 in which the first diluted liquid 21 is stored is formed inside, and the opening of the blood collection tube 201 is hermetically sealed with a sealing body 203 made of, for example, a rubber film. The blood collection tube 201 includes a diluted blood solution storage chamber 20.
The piston 204 for changing the volume of 2 is provided so as to be movable back and forth by rotating a screw-shaped moving back and forth operation rod 205.

On the other hand, the quantitative solution preparation tool 210 has a quantitative solution storage container 211 in which a quantitative solution storage chamber 212 having an open top is formed.
The separation device 213 is attached to the lower part of 11. In this separation device 213, a filter 214 is arranged in a communication holder 215 in which a communication passage is formed, a hollow needle 216 is projectingly arranged below the communication holder 215, and the quantitative solution storage container 211 is arranged. A communication hole 217 communicating with the communication path of the separation device 213 is provided in the lower part of the. The filter 214 holds the cell fraction in the diluted blood solution 22 and allows plasma to pass through.

The method of using such a quantitative solution preparing device is as follows. First, in the case of collecting blood using the blood collecting device 200, as shown in FIG. 19, the advancing / retreating operation rod 205 of the blood collecting device 200 is retracted to increase the volume of the diluted blood solution storage chamber 202, thereby diluting the blood solution. The accommodation chamber 202 is decompressed, and in this state, a hollow needle 206 capable of puncturing the blood sampling part of the human body at both ends.
The hollow needle 206 may be punctured and the other end of the hollow needle 206 may be pierced into the sealing body 203 of the blood collection tool 200. By doing this, the blood flowing out from the blood sampling site
At the same time as being sucked and stored in the diluted blood solution storage chamber 202 in a negative pressure state, the diluted blood solution storage chamber 202 is diluted with the first dilution liquid 21 inside.
In this state, a diluted blood solution is prepared in blood collection tool 200.

Next, the quantitative solution preparing tool 210 will be explained. As shown in FIG. 20, the hollow needle 216 of the separation device 213 is pierced into the sealing body 203 of the blood collecting tool 200 containing the diluted blood solution 22. In this state, the advancing / retreating operation rod 205 of the blood collection tool 200 is advanced to push out the diluted blood solution 22 in the diluted blood solution storage chamber 202 at a positive pressure, thereby the filter 214 of the separation device 213.
The diluted blood solution 22 may be passed through. By doing so, the quantitative solution made of the diluted plasma solution that has passed through the filter 214 is stored in the quantitative solution storage chamber 212 of the quantitative solution storage container 211. The filter 21
In FIG. 4, the cell fraction is captured, and if a quantification solution consisting of a diluted cell fraction solution is prepared, the second dilution is passed through the filter 214 to be captured by the filter 214. The prepared cell fraction may be diluted to prepare a diluted cell fraction solution.

21. Fourth Embodiment FIG. 21 shows a fourth embodiment of a quantitative solution preparing device to which the present invention is applied. In FIG. 21, the quantitative solution preparing device includes a blood collecting tool 300 shown in FIG.
It is composed of a separation device 310 shown in (b) and a quantitative solution containing tube 320 shown in FIG. 21 (c). In the present embodiment, blood collection tool 300 has first blood collection tube 301 inside
The diluted blood solution storage chamber 302 in which the diluted liquid 21 of FIG. It is hermetically sealed with a stopper 303.

On the other hand, in the separation device 310, a filter 312 is arranged in a communication passage in the communication holder 311, and an inlet side hollow needle 313 and an outlet side hollow needle 314 are formed above and below the communication holder 311. . Further, the fixed quantity solution storage pipe 320 has a fixed quantity solution storage chamber 322 formed therein, and the fixed quantity solution storage chamber 322 is vacuumed to a negative pressure in advance, and the opening is made of, for example, a rubber film. It is hermetically sealed with a sealing body 323. In particular, the negative pressure state of the quantitative solution storage tube 320 is set so that the vacuum degree is at least higher than that of the blood collection tube 301 in which the diluted blood solution is prepared.

The method of using such an apparatus for preparing a quantitative solution is as follows. First, in the case of collecting blood using the blood collecting tool 300, as shown in FIG. 22, a hollow needle 306 whose both ends can be punctured is punctured in a blood collecting part of a human body, and the other side of the hollow needle 306 is used as the blood collecting tool. It suffices to pierce the sealing body 303 of 300.
In this way, the blood flowing out from the blood sampling portion is sucked and stored in the diluted blood solution storage chamber 302 in the negative pressure state, and at the same time, diluted with the first diluting liquid 21 inside. In this state, a diluted blood solution is prepared in blood collection tool 300.

Next, the case of preparing a quantitative solution will be described. As shown in FIGS. 23 (a) (b) (c),
The inlet side hollow needle 313 of the separation device 310 is pierced into the sealing body 303 of the blood collection tool 300 containing the diluted blood solution 22, and then the outlet side hollow needle 3 of the separation device 310 is inserted.
14 may be pierced into the sealing body 323 of the quantitative solution containing tube 320. At this time, the quantitative solution storage tube 32
Since the negative pressure state of 0 is higher than that of the blood collection tube 301, the diluted blood solution 22 in the blood collection tube 301 is caused by the pressure difference of the gas.
Passes through the filter 312 of the separation device 310 and is sucked into the quantification solution storage tube 320. As a result, the quantitative solution made of the diluted plasma solution that has passed through the filter 312 is stored in the quantitative solution storage chamber 322 of the quantitative solution storage tube 320. It should be noted that the filter 312 captures the cell fraction, and if a quantification solution consisting of a diluted cell fraction solution is prepared, the filter 312 can be passed through the second diluent to The cell fraction captured in 312 may be diluted to prepare a diluted cell fraction solution.

[Embodiment 5] This embodiment shows a quantification method using the quantification solution prepared in each of the embodiments. This is a method of quantifying a substance to be measured in plasma using blood, which is (1) a step of preparing a quantification solution consisting of a diluted plasma solution by the quantification solution preparation method described above, and (2) From the concentration of the indicator substance in the quantitative solution and the concentration of the indicator substance in the first diluting solution,
Calculating a dilution factor of plasma in the quantitative solution,
(3) a step of measuring the concentration of the substance to be measured in the quantitative solution, (4) the dilution ratio calculated in (2), and the concentration of the substance to be measured in the quantitative solution measured in (3) To the step of quantifying the concentration of the substance to be measured in the plasma (see FIG. 3 (a)). Hereinafter, the quantification method in the present embodiment will be described in detail.

The concentration (X) of the substance to be measured in plasma is the concentration (Y) of the substance to be measured in the quantitative solution prepared by the above two methods, and the dilution ratio of plasma in the quantitative solution. It can be obtained by the equation 1 from (a). X = a × Y (Equation 1) (Method for calculating the dilution ratio of plasma in the quantification solution) The amount of the indicator substance in the first dilution solution is M1, and the volume of the first dilution solution is V1. Then, the concentration C1 of the indicator substance in the first dilution solution is represented by C1 = M1 / V1 (Equation 2). On the other hand, the concentration C2 of the indicator substance in the quantitative solution containing plasma is represented by C2 = M1 / (V1 + V2) (Equation 3) where V2 is the volume of the plasma (however, V2 is not measured). It

The dilution ratio (a) of plasma in the quantitative solution is as follows: dilution ratio (a) = (V1 + V2) / V2 (Equation 4) Therefore, the dilution ratio (a) is C1 and C2. It can be obtained from the value of Dilution ratio (a) = C1 / (C1-C2) (Equation 5) Here, the concentrations C1 and C2 of the indicator substance are absorbance when the indicator substance is a dye or a chromogen, and the indicator substance emits light. When the substance is a substance, it is obtained by measuring the luminescence intensity, and when the indicator substance is a fluorescent substance, it is obtained by measuring the fluorescence intensity. When the indicator substance is quantified by the absorbance, the concentration and the absorbance are proportional to each other. Therefore, the concentration and the absorbance of the indicator substance in the blood diluting solution and the quantifying solution are C1, E1, and C2, respectively.
And E2, C2 / C1 = E2 / E1 (Equation 6) holds. Therefore, the dilution ratio (a) can also be obtained as the dilution ratio (a) = C1 / (C1-C2) = E1 / (E1-E2) (Equation 7).

As described above, the dilution ratio can be calculated from the C1 and C2 values or the E1 and E2 values. Although C1 or E1 may be set to a known value in advance, it can be quantified using a newly prepared first dilution solution. In this case, if a calibration curve showing the relationship between the indicator substance and the information derived from the indicator substance is created, it becomes known.

(Quantitative Method of Indicator Substance) The quantitative method of the indicator substance is not particularly limited as long as it is a method that can quantify the concentration of the indicator substance. When the indicator substance is a dye, the absorbance of the quantification solution itself can be quantified. In other cases, a certain amount of sample is taken out from the quantification solution, and the concentration of the indicator substance in the sample is quantified by an appropriate quantification method of the indicator substance. When the absorbance is used in the quantification, the absorbance value can be directly used without converting into the concentration of the indicator substance. Examples of the method for measuring the concentration of the indicator substance in this example include a colorimetric method, a luminescent method, a fluorescent method, and the like, and the colorimetric method is particularly preferable.

Examples of the indicator used in the colorimetric method include the above-mentioned dyes, reduction-coloring chromogens and oxidation-coloring chromogens. Examples of the oxidative chromogenic chromogen include a leuco chromogen and an oxidative coupling chromogenic chromogen.
As a colorimetric method using the reduced chromogenic chromogen, the reduced chromogenic chromogen is prepared by using a reduced coenzyme such as NAD (P) H, diaphorase and 1-methoxy-5-methylphenadium methylsulfate. The method of converting into a dye by the action of the electron carrier and measuring the absorbance of the produced dye with a spectrophotometer. The colorimetric method using an oxidative chromogenic chromogen is as follows.The oxidative chromogenic chromogen is converted into a dye by the action of hydrogen peroxide and a peroxidase active substance such as peroxidase, and the absorbance of the resulting dye is measured by spectrophotometric analysis. There is a method of measuring with a meter. Examples of the fluorescence method include a method of measuring the fluorescence generated from the above-mentioned fluorescent substance with a peroxide-active substance such as hydrogen peroxide and peroxidase, by a fluorometer.
Examples of the luminescence method include a method in which light (photons) generated from the above-described luminescent substance by a peroxide active substance such as hydrogen peroxide and peroxidase is measured with a luminometer.

When an oxidative coupling chromogenic chromogen is used as the oxidative chromogenic chromogen, one of two compounds that contribute to color development is contained as an indicator substance in the quantitative solution. , The other compound is stored separately.
When the oxidative coloring type chromogen is used as an indicator, the number of moles of the oxidative coloring type chromogen needs to be smaller than the number of moles of hydrogen peroxide. When an oxidative coupling chromogen is used as an indicator, the number of moles of the chromogen needs to be smaller than the number of moles of hydrogen peroxide and the other compound. The hydrogen peroxide used for converting the oxidative chromogenic chromogen to the dye may be hydrogen peroxide itself or may be a substance directly or indirectly produced from a substance by an enzyme. Examples of combinations of substances and enzymes that directly or indirectly generate hydrogen peroxide include the following combinations.

Choline-choline oxidase, cholesterol-cholesterol oxidase, uric acid-uricase, triglyceride-lipoprotein lipase and glycerol oxidase, free fatty acid-acyl-CoA synthetase and acyl-CoA oxidase, glucose-pyranose oxidase, Phospholipid-phospholipase D and choline oxidase, creatine-creatinase and sarcosine oxidase, creatinine-creatininase, creatinase and sarcosine oxidase, lactate-lactose oxidase, inorganic phosphorus-purine nucleoside phosphorylase and xanthine oxidase, Orthotoluoylcholine-cholinesterase and choline oxidase · Monoamines (allylamine) - monoamine oxidase.

(Reagent for calculating dilution ratio) When an oxidative chromogenic chromogen is used as an indicator, the reagent for quantifying the chromogen represents a reagent that converts the chromogen into a dye. The reagent for quantifying the oxidative coloring type chromogen can be stored in one reagent system or a plurality of reagent systems. Storage in a multiple reagent system is preferable, and storage in a two reagent system is more preferable. When hydrogen peroxide itself is used, a two-reagent system that avoids coexistence of hydrogen peroxide and a peroxidase-active substance such as peroxidase is preferable. Further, when hydrogen peroxide is directly or indirectly produced from a substance by an enzyme, a two-reagent system that avoids coexistence of the substance and an enzyme that directly reacts with the substance is preferable.

(Specific Storage Form of Reagent for Calculating Dilution Ratio)
The specific storage form of the reagent for quantifying the oxidative chromogenic chromogen is described below. However, the storage form is not limited to these specific examples. Indicator substance in quantitative solution: EMSE first reagent MOPS buffer (pH 7.1) 20 mmol / L peroxidase 10 U / mL choline chloride 0.05 mg / mL ascorbate oxidase 3 U / mL nonion HS-210 0.1% second Reagent MOPS buffer (pH 7.0) 20 mmol / L 4-aminoantipyrine 0.5 g / L choline oxidase 10 U / mL

(Determination of the substance to be measured in a quantitative solution composed of a diluted plasma solution) The substance to be measured is not particularly limited as long as it is a component in plasma. Is mentioned. The substance to be measured can be quantified by a general method established as a method for quantifying the substance to be measured and is not particularly limited, but a quantification method that is not substantially affected by the indicator substance is preferable. Further, when quantifying a substance to be measured in a quantification solution composed of a diluted plasma solution, the quantification solution may be diluted and used.

Sixth Embodiment This embodiment shows a quantification method using the quantification solution prepared in each of the embodiments. This is a method of quantifying a substance to be measured in a cell fraction using blood, which is a step (1) of preparing a quantification solution consisting of a diluted cell fraction solution by the quantification solution preparation method described above. And (2) calculating a dilution ratio of a cell fraction in the quantification solution from the concentration of the indicator substance in the quantification solution and the concentration of the indicator substance in the second diluent. 3) a step of measuring the concentration of the substance to be measured in the quantitative solution, (4) a dilution ratio calculated in (2),
The step of quantifying the concentration of the substance to be measured in the cell fraction from the concentration of the substance to be measured in the quantification solution measured in (3) (see FIG. 3 (b)). The details of this embodiment are the same as those of the fifth embodiment. ◎ Embodiment 7 This embodiment shows a quantification method using the quantification solution prepared in each of the embodiments. This is a method for quantifying the ratio of hemoglobin A _1C to hemoglobin in a cell fraction using blood, which is (1) a quantification solution comprising a diluted cell fraction solution in the quantification solution preparation method described above. And a step of quantifying the ratio (ratio) of hemoglobin A _1C to hemoglobin in the quantification solution prepared in (2) and (1) (FIG. 3 (c)).
reference).

(Quantification of the substance to be measured in a quantification solution consisting of a diluted cell fraction solution) The substance to be measured is not particularly limited as long as it is a component in the cell fraction, but hemoglobin A _1C is particularly preferable. Preferred examples include: The quantification of the substance to be measured is
It can be carried out by a general method established as a quantitative method of the substance to be measured.

[0087]

EXAMPLES Example 1 (Blood collection tool) The blood collection tool described in Embodiment 1 was prepared (see FIG. 4).
Here, as elements of the blood collecting tool, a suction cup 116 made of polyurethane, a shaft made of polypropylene (link mechanism 142)
A stainless steel 1 mm puncture blade 130 is joined to the polypropylene, a polypropylene rotating plate 141 and two commercially available rubber bands are used as the energy storage member 160.
The tool body 110 is made of a transparent material so that it can be observed from the outside when blood flows in using an acrylic resin. The piston portion 181 of the pressure reducing mechanism 180 is made of polypropylene. Further, an operation handle 153 is externally coupled to the rotary plate 141, and when the operation handle 153 is rotated, the rotary plate 141 rotates to stretch the rubber, and energy is stored.

Example 2 Preparation of Quantitative Solution Consisting of Diluted Plasma Solution Using Blood Collection Tool According to Example 1 (1) Blood Collection In FIG. 4, the external operation handle 153 to which the rotary plate 141 is connected is The rubber of the energy accumulating member 160 was stretched to accumulate energy, and the engaging / disengaging mechanism 170 stopped the puncture blade 130 so as to return to the initial position. As a blood sampling portion of a human body, for example, the suction cup 116 is pressed against the inside of the arm to adsorb the suction cup 116, and the locking rod 177 is pushed out to release the locking state between the locking member 171 and the rotary plate 141. Puncturing operation was performed by the puncture blade 130 by rotating all at once, and blood was collected at the same time. After one minute, the suction cup 116 was removed, the operation handle 153 was rotated in the reverse direction, and blood was pushed out. The obtained blood was about 0.2 mL.

(2) Preparation of Quantitative Solution Consisting of Diluted Plasma Solution The blood collected in (1) was diluted with the following blood-diluting solution (first diluent), and the resulting diluted blood solution was used as an acrylic resin. Separated with adhesive glass fiber filter paper (Millipore),
A quantitative solution consisting of a diluted plasma solution was prepared. The quantification solution contains glycerol-3-phosphate as an indicator substance. PIPES buffer (pH 7.0) 50 mmol / L heparin 10 kU / L sodium chloride 5 g / L α-ketoglutarate 1.5 g / L glycerol-3-phosphate 0.5 g / L sodium azide 0.3 g / L

Example 3 (quantitative solution preparation instrument) This example is a model according to the third embodiment and is an instrument for preparing a quantitative solution consisting of a diluted plasma solution,
A device having a function of collecting blood and preparing a diluted blood solution (a blood collecting device), and a device having a function of preparing a quantitative solution composed of a diluted plasma solution (a quantitative solution preparing tool: sometimes referred to as a cell fraction separating tool) There is). The device having the functions of blood collection and diluted blood solution preparation is
It has a blood collecting function, a diluted blood solution preparation function, and a diluted blood solution accommodation function. The device having the function of preparing a quantitative solution composed of a diluted plasma solution has a function of preparing a quantitative solution composed of a diluted plasma solution and a function of accommodating a quantitative solution composed of a diluted plasma solution. The quantitative solution preparation instrument consisting of these two tools is a device for collecting blood and diluting it at the same time to separate the diluted blood solution into blood cells, and is mainly made of polypropylene. That is, the main body is polypropylene and the sealing body is rubber made of polybutadiene. Further, as the filter, a glass filter using an acrylic resin having a diameter of micron as a binder is laid in a thickness of 5 mm. 0.5 mL of the first diluting liquid having the same composition as the first diluting liquid used in Example 2 was placed in the blood collecting tool and sealed with a sealing body made of a rubber-like film.

Example 4 Preparation of Quantitative Solution Consisting of Diluted Plasma Solution Using Quantitative Solution Preparation Device of Example 3 (1) Blood Collection and Preparation of Diluted Blood Solution First, the operation advancing / retreating rod in the blood collecting tool of Example 3 Was rotated to move the piston to the side opposite to the sealing body made of a rubber-like film to generate a reduced pressure. Using a commercially available vacuum blood sampling adapter (made by Terumo) that has needles at both ends, pierce the arm with the needle and penetrate the needle on the side opposite to the arm into the sealing body made of rubber-like film I had my blood sucked. After 30 seconds, the needle was removed from the arm, and the needle was also removed from the sealed body of the blood collection tool. Blood entered the inside of the blood collection tool and was mixed with the first diluent to prepare a diluted blood solution. (2) Preparation of Quantitative Solution Consisting of Diluted Plasma Solution Next, in order to mount the quantitative solution preparing tool (cell fraction separating tool) of Example 3, the sealing body made of a rubber-like membrane of the blood collecting tool is hollow. The needle was pierced, and the blood collection tool and the quantitative solution preparation tool were attached by, for example, screwing. In this state, make the whole of the fixed amount solution container of the fixed amount solution preparation device vertical so that it is in the vertical direction, and then turn the operation advancing / retreating rod in the opposite direction to decrease the volume of the diluted blood solution storage chamber. Moved the piston. Then, the blood collection tool is pressurized, the diluted blood solution is filtered by a filter, the cell fraction is captured by the filter, and the quantitative solution consisting of a slightly yellowish diluted plasma solution is stored in the quantitative solution storage container. It was

Example 5 Preparation of Quantitative Solution Consisting of Diluted Plasma Solution Using Quantitative Solution Preparation Device This Example uses the quantification solution preparation device according to the fourth embodiment, and Blood was collected with a vacuum blood collection tube containing a diluent and a diluted blood solution was prepared in the blood collection tube. The needle portion of the separation device was inserted into the blood collection tube containing this diluted blood solution. The separating device has hollow needles mounted on both sides, and the needles on both sides are connected through a central filter. As the filter, acrylic resin-bonded glass fiber filter paper (manufactured by Millipore) was used.
Next, a vacuum blood collection tube capable of collecting blood of about 1 to 2 mL is inserted into the needle on the opposite side, and the diluted blood solution in the upper portion is filtered through a filter by depressurization, and a quantitative solution consisting of the diluted plasma solution in the lower vacuum blood collection tube. was gotten.

Example 6 Determination of Uric Acid in Plasma Using the Quantitative Solution Consisting of the Diluted Plasma Solution Prepared in Example 4 (1) Calculation of Dilution Ratio In the quantifying solution comprising the diluted plasma solution prepared in Example 4 The dilution ratio of plasma was calculated by measuring the absorbance using the following reagent for calculating the dilution. The absorbance was measured using an automatic analyzer Toshiba 200FR with the following measurement parameters. As a result, the dilution ratio of plasma in the quantitative solution was calculated to be 12.8. Solution for Dilution Calculation First Reagent PIPES Buffer (pH 7.0) 20 mmol / L Triton X-100 1.0 g / L EMSE 0.2 g / L Sodium azide 0.3 g / L Ascorbate oxidase 1 U / mL Peroxidase 5U / ML Second reagent PIPES buffer solution (pH 7.0) 50 mmol / L Triton X-100 1.0 g / L 4-aminoantipyrine 0.5 g / L sodium azide 0.3 g / L peroxidase 10 U / mL glycerol-3- Phosphate oxidase 20 U / mL Reagent parameter for calculating dilution ratio Sample volume: 5 μL R1: 150 μL R2: 50 μL Main wavelength 546 nm Sub-wavelength 700 nm Photometric point: (14-16) (31-33)

(2) Measurement of Uric Acid Concentration in the Quantitative Solution The concentration of uric acid in the quantified solution consisting of the diluted plasma solution prepared in Example 3 was measured by the uric acid measuring reagent Determiner L.
It was measured using R1 and R2 of UA (Kyowa Medex Co., Ltd.). The measurement was performed using an automatic analyzer Toshiba 200FR with the following measurement parameters. as a result,
The uric acid concentration in the quantitative solution was 0.27 mg / dL. Measurement parameters for uric acid measurement Sample volume: 15 μL R1: 150 μL R2: 50 μL Main wavelength 546 nm Sub wavelength 700 nm Photometric point: (14-16) (31-33)

(3) Determination of Uric Acid in Plasma From the dilution ratio of 12.8 calculated in (1) and the concentration of uric acid in the quantitative solution measured in (2) of 0.27 mg / dL, The uric acid concentration was quantified as 3.5 mg / dL. On the other hand, separate blood (5 mL) was collected from a blood donor used in Example 3 into a heparin-containing test tube, and the heparin-containing blood was centrifuged (3000 rpm, 10 minutes),
Plasma was obtained. The concentration of uric acid in the plasma was determined as described above.
Uric acid measurement reagent Determined using R1 and R2 of Determiner L UA (manufactured by Kyowa Medex Co., Ltd.). As a result, the concentration of uric acid in the plasma was 3.6 mg / dL, which is extremely close to the concentration of uric acid of 3.5 mg / dL determined by the method of the present invention. It was found that the method for quantifying uric acid described above was comparable to the conventional method for quantifying uric acid.

[0096]

As described above, according to the blood collecting device of the present invention, a system for automatically puncturing a puncture body by utilizing accumulated energy is adopted and a predetermined amount of blood is stored in the blood storage chamber. Since blood can be collected by suction, it is possible to eliminate mental pain when the puncture body is punctured by itself and to collect a certain amount of blood easily and reliably.
According to another aspect of the present invention, there is provided a blood collecting device, a device main body in which a first diluting liquid is stored, and a hermetically sealed diluting blood solution storage chamber is formed, and the diluting blood. Since the puncture body connected to the solution storage chamber is provided, the diluted blood solution can be easily generated at the same time as blood collection. In short, in any aspect of the present invention,
Since it is easy to collect blood or prepare a diluted blood solution, it is possible to easily prepare a quantitative solution.

Further, in the method for preparing a quantitative solution according to the present invention, after diluting the collected blood, it is separated into plasma and cell fractions by a separating means, and comprises a diluted plasma solution or a diluted cell fraction solution. Since the quantification solution was prepared, a quantification solution consisting of a diluted plasma solution or a diluted cell fraction solution can be prepared quickly, simply, and safely using the diluted blood solution collected and prepared. Is possible.
Therefore, it becomes possible to quantify the substance to be measured in plasma and the substance to be measured (particularly, hemoglobin A _1C ) in the cell fraction using a solution for quantification consisting of a diluted plasma solution or a diluted cell fraction solution. Further, according to the quantitative solution preparing device of the present invention, the above-mentioned quantitative solution can be easily prepared.

[Brief description of drawings]

FIG. 1A is an explanatory diagram showing an outline of a blood collecting device according to the present invention, and FIG. 1B is an explanatory diagram showing an outline of a blood collecting device according to another aspect of the present invention.

FIG. 2A is an explanatory diagram showing an outline of a method for preparing a quantitative solution according to the present invention, and FIG. 2B is an explanatory diagram showing an outline of an instrument for preparing a quantitative solution according to the present invention.

FIG. 3 (a) is an explanatory diagram showing a quantification method using a quantification solution comprising a diluted plasma solution according to the present invention, and (b) and (c) are quantifications comprising a diluted cell fraction solution according to the present invention. It is an explanatory view showing a quantification method using a solution for use.

FIG. 4 is a first embodiment of a blood collecting device to which the present invention is applied.
It is sectional explanatory drawing which shows.

5 is an explanatory side view of FIG.

6A is an explanatory diagram showing a housing of the blood collecting tool according to the first embodiment, and FIG. 6B is an explanatory diagram showing a piston portion.

7A is a front view and a plan view showing a link mechanism that supports a puncture blade, FIG. 7B is a view seen from the direction B in FIG. 7A, and FIG. 7C is C in FIG. 7A. The front part which shows the detail of a part, and its left side view, (d) is the front view which shows the detail of D part in (a), and its left side view.

FIG. 8 is an explanatory diagram showing a diluent storage container used in the first embodiment.

9A is a front view and a left side view of the rotation drive device used in the first embodiment, and FIG. 9B is a front view of a locking member of an engagement / disengagement mechanism that engages / disengages with the rotation drive device. It is a left side view.

10A is a front view and a right side view of a rod attachment portion of an engagement / disengagement mechanism that engages / disengages with a rotation drive device, and FIG. 10B is an explanatory view showing a locking rod of the engagement / disengagement mechanism.

FIG. 11 is an explanatory diagram showing an operation process (1) of the blood collection device according to the first embodiment.

FIG. 12 is an explanatory diagram showing an operation process (2) of the blood collection tool according to the first embodiment.

FIG. 13 is an explanatory diagram showing an operation process (3) of the blood collection device according to the first embodiment.

FIG. 14 is an explanatory diagram showing a modified form of the blood collection tool according to the first embodiment.

15 (a) and 15 (b) are explanatory views showing a configuration other than a blood sampling tool of the quantitative solution preparing instrument used in the second embodiment.

16 (a) to 16 (c) are explanatory views showing a usage method (1) of the quantitative solution preparing instrument used in the second embodiment.

17 (a) to 17 (c) are explanatory views showing a usage method (2) of the quantitative solution preparing instrument used in the second embodiment.

18 (a) and 18 (b) are explanatory views showing a quantitative solution preparing device according to a third embodiment.

FIG. 19 is an explanatory diagram showing a blood sampling method according to the third embodiment.

FIG. 20 is an explanatory diagram showing a method for preparing a quantification solution in the third embodiment.

21 (a) to 21 (c) are explanatory views showing a quantitative solution preparing device according to a fourth embodiment.

FIG. 22 is an explanatory diagram showing a blood sampling method according to the fourth embodiment.

23 (a) to 23 (c) are explanatory views showing a method for preparing a quantitative solution according to the fourth embodiment.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Tool body, 2 ... Blood storage chamber, 3 ... Blood suction part, 4 ...
Blood collection part, 5 ... Puncture body, 6 ... Advance / retreat moving mechanism, 7 ... Engagement mechanism, 8 ... Energy storage means, 9 ... Decompression mechanism, 10 ... Diluting liquid storage chamber, 11 ... Tool body, 12 ... First dilution Liquid, 13
... diluted blood solution storage chamber, 14 ... volume variable mechanism, 15 ... puncture body, 16 ... diluted blood solution, 17 ... quantitative solution (diluted plasma solution), 18 ... second diluent, 19 ... quantitative solution (diluted Cell fraction solution), A ... Blood collection tool, B ... Separation means, C ...
Solution container for quantitative determination

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61B 5/14 300C 310 (72) One inventor Miyauchi, Kamiwachi 600 No. 1 Kamiyamachi, Nagaizumi-cho, Sunto-gun, Shizuoka Prefecture F-Term inside the Kyowa Medex Research Center, Medex Co., Ltd. (reference) 2G045 AA13 BB04 BB05 BB41 CA01 CA25 CA26 DA48 DA51 FA07 HA06 HA14 4B029 AA09 BB11 HA05 4C038 KK00 KL01 KL09 KM00 KY03 KY04 TA01 TA04 TA10 UA10 UG10 UC10 UG10 UB10 UG

Claims (23)

[Claims] 1. A tool main body having a blood storage chamber formed therein and having a blood suction portion communicating with the blood storage chamber for keeping a tight seal with a blood sampling portion, and a retractable body with respect to the tool main body. A puncture body for puncturing the blood sampling portion, an advance / retreat movement mechanism for moving the puncture body forward / backward between an initial position or a retracted position and a puncture position, and a puncture body detachable at the initial position. The engagement / disengagement mechanism that locks the puncture body, the energy storage means that stores the driving energy required for the puncture body to advance at least from the initial position to the puncture position, and transmits it to the advancing / retracting movement mechanism. A blood collecting device, comprising: a decompression mechanism for decompressing a blood storage chamber to a negative pressure state in association with an advancing / retreating movement mechanism in a process of retreating to a position. 2. A blood accommodating chamber is formed inside, and has a blood suction part which communicates with said blood accommodating chamber and maintains a tight seal with a blood sampling part, and said blood accommodating chamber has a first diluent. And a puncture body for puncturing the blood sampling part, which is provided so as to advance and retreat with respect to the tool body, and a puncture body for puncturing the blood sampling part. Between the position and the puncture position, an advancing / retreating mechanism for moving the puncture body forward and backward, an engagement / disengagement mechanism for releasably locking the puncture body at the initial position, and Energy storage means that stores the required driving energy in the forward and backward movement mechanism, and the puncture body moves backward from the puncture position to the retracted position in the process of moving backward and forward, moving the forward and backward movement mechanism to reduce the pressure of the blood chamber to a negative pressure state. Equipped with a decompression mechanism to Blood collection tool that the butterflies. 3. The blood collecting device according to claim 1, wherein the energy storage means drives the puncture body to advance from the initial position to the puncture position and to retract from the puncture position to the retracted position. A blood collecting tool characterized by accumulating energy. 4. The blood collection tool according to claim 1, wherein the decompression mechanism includes a piston portion that advances and retracts the blood storage chamber in association with the forward and backward movement of the puncture body by the forward and backward movement mechanism, and the puncture by the forward and backward movement mechanism. The blood storage chamber is decompressed in conjunction with the retreat operation of the body, and the blood in the blood storage chamber is pushed out in conjunction with the re-advancing operation of the puncture body from the retracted position to the puncture position by the advancing / retreating mechanism. Blood collection tool. 5. The blood collecting device according to claim 1, wherein the puncture body advance / retreat mechanism can be moved forward / backward by a manual operation. 6. A first diluent is contained inside, and
A device body in which a diluted blood solution storage chamber is formed in a sealed and depressurized state, and a puncture body that is connected to the diluted blood solution storage chamber of the device body and pierces a blood sampling portion to guide blood to the diluted blood solution storage chamber. A blood collection device characterized by comprising: 7. A first diluent is contained inside, and
A device body having a sealed variable blood solution storage chamber in a reduced pressure state and a variable volume mechanism for varying the volume of the sealed diluted blood solution storage chamber, and a device for communicating with the diluted blood solution storage chamber of the device body A blood sampling device, comprising: a puncture body that is connected and that punctures a blood sampling portion to guide blood to a diluted blood solution storage chamber. 8. A method for preparing a quantification solution comprising a diluted plasma solution for quantifying a substance to be measured in plasma, using the collected blood, which is (1) according to claim 1 or 2. Using the blood collecting tool, collecting blood in the blood chamber, and diluting the collected blood with a first diluent containing an indicator substance of known concentration, and (2) this diluted blood solution, A method for preparing a quantitative solution, comprising the step of separating a plasma fraction and a cell fraction by a separating means to prepare a diluted plasma solution. 9. A method for preparing a quantification solution comprising a diluted cell fraction solution for quantifying a substance to be measured in a cell fraction, using the collected blood, which comprises (1). Or, using the blood collecting tool described in 2 above, collecting blood in the blood containing chamber, and diluting the collected blood with the first diluent, and (2) converting the diluted blood solution into plasma by separating means. A solution for quantification, comprising: a step of separating into a cell fraction; and (3) a step of diluting the separated cell fraction with a second diluent to prepare a diluted cell fraction solution. Preparation method. 10. A diluted cell fraction for quantifying the ratio of hemoglobin A _1C to hemoglobin in a cell fraction, which comprises a diluted cell fraction solution for quantifying a substance to be measured in the cell fraction. The method for preparing a quantitative solution according to claim 9, which is a quantitative solution comprising a separate solution. 11. A method for preparing a quantification solution comprising a diluted plasma solution for quantifying a substance to be measured in plasma using the collected blood, which is (1) according to claim 6 or 7. A step of generating a diluted blood solution in the diluted blood solution storage chamber using a blood sampling tool; and (2) the diluted blood solution,
A method for preparing a quantitative solution, comprising the step of separating a plasma fraction and a cell fraction by a separating means to prepare a diluted plasma solution. 12. A method for preparing a quantification solution comprising a diluted cell fraction solution for quantifying a substance to be measured in a cell fraction, using the collected blood, which comprises (1).
Alternatively, using the blood collecting tool described in 7, collecting blood into the blood chamber and diluting the collected blood with a first diluent, (2) converting the diluted blood solution into plasma by separating means. A solution for quantification, comprising: a step of separating into a cell fraction; and (3) a step of diluting the separated cell fraction with a second diluent to prepare a diluted cell fraction solution. Preparation method. 13. A diluted cell fraction for quantifying a ratio of hemoglobin A _1C to hemoglobin in a cell fraction, which comprises a diluted cell fraction solution for quantifying a substance to be measured in the cell fraction. The method for preparing a quantitative solution according to claim 12, which is a quantitative solution comprising a partial solution. 14. The blood collecting device according to claim 1 or 2, and a separation means for producing a diluted blood solution from blood collected by the blood collecting device and separating plasma and cell fraction from the diluted blood solution. And a quantification solution preparation container for accommodating a quantification solution accommodating container, which comprises the diluted plasma solution separated and prepared by the separation means. 15. A blood collection tool with a variable volume mechanism according to claim 7, separation means for separating plasma and cell fraction from a diluted blood solution collected and prepared by the blood collection tool, and the separation means. A quantification solution preparation instrument, comprising: a quantification solution container for accommodating a quantification solution consisting of the separately prepared diluted plasma solution. 16. The quantification solution preparation device according to claim 15, wherein the separating means is provided at an inlet portion of the quantification solution storage container, holds a cell fraction and allows plasma to pass, and the filter. And a communication member that is provided on the inlet side and is connected to communicate with the diluted blood solution storage chamber of the blood collection tool, and reduces the volume of the diluted blood solution storage chamber by the volume variable mechanism, thereby diluting the diluted blood solution storage chamber. A quantification solution preparation instrument, characterized in that a quantification solution comprising a diluted plasma solution is prepared in a quantification solution storage container by passing a blood solution through the separating means. 17. The method for preparing a solution for quantification according to claim 16, wherein the blood collecting tool according to claim 7 is used to generate a diluted blood solution in the diluted blood solution storage chamber, and then the blood collecting tool is used. After connecting the communication member of the separation means to the diluted blood solution storage chamber, reduce the volume of the diluted blood solution storage chamber by the volume changing mechanism of the blood collection tool, and pass the diluted blood solution in the diluted blood solution storage chamber to the separation means. A method for using a quantification solution preparation instrument, comprising preparing a quantification solution consisting of a diluted plasma solution in a quantification solution storage container. 18. The blood collecting tool according to claim 6, further comprising a tool main body in which the first diluting liquid is housed, and a diluting blood solution housing chamber which is hermetically sealed and depressurized is formed in advance. The separation means for separating plasma and cell fractions from the diluted blood solution collected and prepared by A quantitative solution preparing instrument for storing a quantitative solution containing a quantitative solution comprising a diluted plasma solution separated and prepared by means. 19. The device for preparing a solution for quantification according to claim 18, wherein the separating means is a filter that holds a cell fraction and allows plasma to pass through, and the diluted blood solution storage chamber that is provided on the inlet side of the filter. And an outlet-side communication member that is provided on the outlet side of the filter and that is connected to the quantitative solution storage chamber, the diluted blood solution in the diluted blood solution storage chamber of the blood collecting device. Is passed through the separating means to prepare a quantitative solution comprising a diluted plasma solution in a quantitative solution storage container. 20. When using the device for preparing a quantification solution according to claim 19, a tool main body in which the first diluting solution is housed, and a sealed diluting blood solution housing chamber is previously formed. 7. The blood sampling device according to claim 6 is used, and a diluted blood solution is generated in the diluted blood solution storage chamber, and after that, an inlet side communication member of the separating means is connected to the diluted blood solution storage chamber of the blood sampling device. , The outlet side communication member of the separating means is connected to the fixed amount solution storage chamber of the fixed amount solution storage container, and the diluted blood solution storage of the blood collection device is performed by the pressure difference between the fixed volume solution storage chamber and the diluted blood solution storage chamber. A method for using a quantification solution preparation instrument, characterized in that a quantification solution comprising a diluted plasma solution is prepared in a quantification solution storage container by passing a diluted blood solution in a room through the separation means. 21. A method for quantifying a substance to be measured in plasma using blood, comprising: (1) preparing a quantification solution comprising a diluted plasma solution by the method according to claim 8 or 11; (2) calculating a dilution ratio of plasma in the quantification solution from the concentration of the indicator substance in the quantification solution and the concentration of the indicator substance in the first diluent, and (3) the quantification From the step of measuring the concentration of the substance to be measured in the solution for use, the dilution ratio calculated in (4) and (2), and the concentration of the substance to be measured in the solution for quantification measured in (3) And a step of quantifying the concentration of the substance to be measured. 22. A method for quantifying a substance to be measured in a cell fraction using blood, which comprises (1) preparing a quantification solution comprising a diluted cell fraction solution by the method according to claim 9 or 12. And (2) calculating a dilution ratio of the cell fraction in the quantification solution from the concentration of the indicator substance in the quantification solution and the concentration of the indicator substance in the second diluent. , (3)
Measuring the concentration of the substance to be measured in the quantitative solution,
(4) A step of quantifying the concentration of the substance to be measured in the cell fraction from the dilution ratio calculated in (2) and the concentration of the substance to be measured in the quantification solution measured in (3). A quantification method comprising: 23. A method for quantifying the ratio of hemoglobin A _1C to hemoglobin in a cell fraction using blood, which comprises (1) quantifying a diluted cell fraction solution by the method according to claim 10 or 13. Preparing a solution for use,
(2) A step of quantifying the ratio of hemoglobin A _1C to hemoglobin in the quantification solution prepared in (1), which is a quantification method.