JP3314060B2 - Automatic sampling system for biological fluids - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biological fluid sampling apparatus for automatically collecting blood and other biological fluids from animals.

[0002]

2. Description of the Related Art In animal experiments, in order to examine, for example, pharmacokinetics and biological rhythm, blood must be repeatedly collected from the animal at regular intervals, and this is generally a manual operation. If the experiment lasts for a long time, the blood collection work often extends from midnight to early morning, which places a great burden on the person in charge. In addition, the approach of humans at each blood sampling causes stress on animals and affects the state of blood. This is the same when other biological fluids such as urine and bile are collected.

Conventionally, as one of the few attempts to automate the blood collection operation, a three-way solenoid valve is used, and a blood collection means such as a catheter embedded in a rat body is connected to the tip of a flow path extending from one port, A blood storage device such as a fraction collector is connected to a tip of a flow path extending from another one port, and a washing liquid container made of heparin-containing saline is connected to a tip of a flow path extending from the other one port,
There is a system in which a reversible peristaltic (ironing) pump is inserted in the middle to repeatedly perform blood collection / storage, cleaning of three channels including a solenoid valve, and standby. However, this method has the following drawbacks in terms of mechanism and flow path configuration.

First, the three-way solenoid valve has a complicated internal structure consisting of a three-way port and a valve body for selectively communicating between the three-way port, so that a small-volume washing liquid has a high viscosity and tends to solidify. It is difficult to wash out the blood and a large amount of washing liquid has to be flushed. This large amount of washing solution is eventually sent into the animal body and affects the next blood sample. Conversely, if the amount of the washing liquid is small, blood remains in the solenoid valve, solidifies and blocks the flow path system, so that normal blood collection cannot be continued.

[0005] Second, in this method, blood from the animal body and the washing liquid come into interface with each other, causing blood diffusion. If the diluted blood area spreads as a result, a larger amount of cleaning liquid is required for cleaning the expanded area, and all the cleaning liquid is returned to the animal body, and the effect on the animal is serious.

Third, in the inversion drive of the peristaltic pump, it is difficult to control the amount of blood collected, and a small amount of blood cannot be collected with good reproducibility.

[0007]

SUMMARY OF THE INVENTION As described above, the present invention overcomes the drawbacks of the conventional automatic blood sampling apparatus, which is hardly practical, and ensures accurate and reproducible over a long period of time without manual work. It is an object of the present invention to provide a device that can automatically and quantitatively collect blood from experimental animals.

[0008]

In order to achieve the above object, the present invention relates to an apparatus for automatically collecting biological fluid of an animal at an arbitrary interval, wherein five ports of a five-way joint are connected to five tubes, respectively. At the same time, the connection bases of at least four of the tubes can close the flow path by the bending deformation of the tube itself, and the tip of the first tube connected to the first port is embedded in the animal body. The tip of the second tube connected to the second fluid port is connected to the second port, and the tip of the second tube connected to the second port is opened as an intake port, and the tip of the third tube connected to the third port is controlled by the biological fluid sample. The tip of the fourth tube connected to the actuated fractionating means and connected to the fourth port is opened as a waste liquid port, and the tip of the fifth tube connected to the neutral port is connected to the control actuated syringe. Along with Immediately before the syringe, it is connected to the branch flow path to the washing liquid reservoir, and the connection bases of the first to fourth tubes are selectively connected in accordance with time-series control related to each control operation of the syringe and the sorting means. By bending and deforming the flow path and closing the flow path, a sampled fraction from the moving body divided into front and rear by a gas gap sucked and formed from the intake port is formed in the fifth tube, and then the third is formed.
And a gas gap is formed in a portion of the first tube immediately before the biological fluid collecting means in a standby state, and four gas gaps excluding the gas gap and the second tube are provided. An automatic sampling apparatus for biological fluid is provided which fills a 5-way joint flow path including a tube and a 4-way port corresponding to the inside of a connection base with the washing solution.

[0009] According to the above device, the opening / closing and switching portion of the flow channel, which is generally considered to be most likely to retain blood in the flow channel, is not a cock type but a flexible tube-shaped connection base. Specifically, by sandwiching from the outside, the flow passage is closed by causing a bending deformation such that the cross section becomes flat, so that there is no step on the flow passage forming surface (the inner surface of the connection base) through the opening and closing process. The continuity is maintained, and the blood circulation can be improved and the residue can be eliminated.

A gas suction tube having a suction port at the tip is connected to the five-way joint of the apparatus as one of the flow paths unique to the present invention. By interposing a gas gap between a biological fluid such as that described above and a washing fluid, these two liquid phases are prevented from contacting and diffusing with each other. Therefore, there is almost no region where the biological fluid and the cleaning fluid are mixed, the biological fluid collected from the animal is minimized, and further, the cleaning fluid exhibits an excellent effect that only a very small amount mixed with the biological fluid is injected into the animal. .

[0011] Further, a waste liquid tube having a waste liquid port at the tip thereof is connected to the five-way joint as another flow path unique to the present invention, and the cleaning liquid that has washed residual blood in the joint portion can be directly discharged. Therefore, it is possible to minimize the amount of washing liquid in the animal-side flow path.

In order to detect the occurrence of a thrombus at the distal end of a sampling catheter implanted in an animal body or the occurrence of clogging in a flow path system, a pressure sensor is provided in a flow path close to a push-pull syringe. Install. Thus, when clogging occurs, the pressure sensed by the sensor increases during the push stroke and decreases during the pull stroke, so that such pressure changes can be monitored to control the operation of the device.

[0013]

FIG. 1 is a perspective view showing the overall configuration of a four-channel automatic sampling apparatus for a biological fluid, in this case, animal blood, of the present invention. In FIG. 1, the apparatus includes an operator operation section 1 protruding from the front of the apparatus, a push-pull syringe section 2 located below the operation section, a blood storage section 3 behind them and forming the center of the apparatus, and further behind the apparatus. , An X-axis driving section 4X, a blood sampling flow path section 5, and experimental animal sections 6a, 6b, 6c and 6d. The X-axis drive unit 4X supports a Y-axis drive unit 4Y having four hanging spouts 7 so as to be movable in the X-axis direction (the left-right direction in the figure). The animal is located below the joint boxes 5a, 5b and 5c, 5d of each channel located above each part corresponding to the four experimental animal parts 6a to 6d. The Y-axis drive unit 4Y
While moving in the axial direction (the front-back direction of the apparatus), the four dropping outlets 7 are moved up and down to sequentially inject the blood collection fraction into each vial of the sample vial row assigned to each channel in the blood storage unit 3. It is to let.
In FIG. 1, the Y-axis driving unit 4Y is located on the washing table 3a (home position) connected to the left end of the blood storage unit 3, and each injection port corresponds to four spouts opened on the same table 3a. ing.

Rats, cats, dogs and the like are used as experimental animals, and a catheter for blood collection is implanted in these animals in advance and operated. For recovery, the animals were reared for several days after the operation, and then the box-shaped experimental animal parts 6a to 6d
It is housed under anesthesia or in an unrestrained state. In each of the joint boxes 5a to 5d, a five-way joint is housed, and the tip of the first tube 5 connected to the first port of each of the five-way joints hangs down in each animal breeding unit, and the catheter described above. And the like. 5 each
The second port to the fifth port of the two joints are respectively provided with a gas source such as air or carbon dioxide gas, a driving unit 4Y which constitutes a control-operated type fractionating means for a blood sample, and a drain fluid drain channel (not shown). ), And four tubes respectively led to four syringes in the push-pull syringe part 2. The blood storage unit 3 can be cooled down to -8 ° C in order to prevent the collected blood from deteriorating.

The basic specifications of this four-channel device are, for example, a sampling amount: 10 to 150 μl, a sample holding capacity: 16 rows × 16 = 256, a number of fractions: 1,
208 samples for 2 animals, 112 samples for 2 animals, 64 samples for 3 animals, 648 samples for 4 animals, and sampling interval can be set in the range of 10 minutes to 18 hours,
It can be cooled from room temperature to −8 ° C. at an ambient temperature of 25 ° C., and is driven by a rated current of 10 A by connecting to an AC 100 V commercial power supply.

FIG. 2 shows each joint box 5a of FIG.
It is a perspective view which shows the five-way joint accommodated in each of-5d, and its connection state. The five-way joint 8 is provided with a four-way flow passage extending radially from the center of a cylindrical body made of a suitable synthetic resin or the like.
And 8d are connected to each other, and a neutral flow path extending in the axial direction from the central node of the four-way flow path, that is, a fifth flow path, is connected to the neutral port 8e. In the first port 8a to the fourth port 8d, and in this case, also in the neutral port 8e, these ports have tube connection bases 9a to 9e made of silicone rubber.
Are respectively mounted. An extension tube represented by an arrow or a solid line is inserted and connected to each of the distal end openings of these connection bases, but the tube of each connection base may be extended as it is. The connection bases, particularly the connection bases 9a to 9d for the four-way opening, are bent and deformed into a flat shape by being squeezed from a direction orthogonal to each axis, and constitute a pinch valve for closing the lumen flow path. ing. 4way 8a
The destination of the tube connected to ８8d is as described above, but the distal end of the tube extending from the neutral port 8e is connected to the syringe 11 in the syringe section 2 and the physiological saline containing heparin via the solenoid valve 10. A pressure gauge 13 is disposed in front of the solenoid valve 10 and connected to the washing liquid reservoir 12 for controlling the apparatus.

The flow path configuration of each five-way joint is as described above. This operation sequence will be described in detail below with reference to flow path schematic diagrams shown in FIGS. In these figures, each connection base 9a to 5
The 9d pinch valve functions are 1), 2), 3) and 4), respectively.
And their open state is a white rectangle,
In addition, the closed state is indicated by inserting a cross in a rectangle. First, FIG. 3A shows a standby state, in which the four-way pinch valves are all closed, and the pinch valve 2) closed from the center node of the five-way joint 8 and the suction port at the tip of the tube extending from the pinch valve. All the channels are substantially filled with the cleaning liquid except that up to 14 is filled with a gaseous phase such as air or carbon dioxide gas. Overall 6
The white area b in the tube close to the animal 15 in the animal part is a gas gap for interrupting the washing liquid.

When the sequence is started, as shown in FIG. 3B, first, the gas channel pinch valve 2) is opened, and the syringe 11 is pulled (suctioned). (Hereinafter, the operation of the syringe 11 is assumed to be pulling if the arrow along the tube 16 guided to it is rightward in the figure, and push (push) if leftward). A cleaning gas gap a is formed from the central node of the joint 8 to the tube 16. Next, in the same pull state, as shown in FIG. 3C, a pinch valve 2)
Is closed, and the pinch valve 1) connected to the tube 7 guided to the blood collecting means in the animal body is opened. As a result, blood is drawn from the animal, and a blood collection fraction connected to the gas gap b drawn into the tube 16 is formed. The leading end of this fraction is depicted in the figure as a completely empty thick line representing the fullness of blood, with a gradually increasing black spot distribution indicating dilution.

FIG. 4 shows a state in which a blood collection fraction for a sample is formed. As shown in FIG. 4A,
In the continuous pull state of the syringe, only the pinch valve 2) of the gas introduction path is opened, and a gas gap c is formed at the rear end of the first blood collection fraction in the tube 16.
Next, as shown in FIG. 4B, only the pinch valve 1) of the blood collection flow path is opened while the pull state is maintained, and the blood collection flow from the animal 15 following the gas gap c passes through the central node of the five-way joint 8. Formed through Further, as shown in FIG. 4C,
In the pull state, only the pinch valve 2) is opened, and a gas gap d at the rear end of the second blood fraction following the gas gap c is formed.

Next, as shown in FIG. 5A, the syringe
The push operation is performed, and the pinch valve 3) at the base of the tube 17 reaching the blood storage unit is opened.
As a result, the second blood collection fraction having substantially no dilution portion between the gas gaps dc and
The blood collection fraction that has been introduced into the tube 16 and that has previously been introduced into the tube 16 is returned to a position where the rear end is partitioned by the gas gap c that straddles the central node of the five-way joint 8. Further, as shown in FIG. 5B, in the syringe push state, the pinch valve 4) of the waste liquid flow path is opened, and the waste liquid is prevented so that the part of the gas gap c other than the blood storage side (upper side in the figure) does not enter the animal body. It is turned to the mouth. Next, in FIG. 5c, the pinch valve on the animal side
1) is opened and the other gas gap b of the first blood collection fraction is
The blood is returned to the animal until it enters the side mouth.

Next, as shown in FIG. 6A, the pinch valve 3) to the blood storage unit is opened in the push state, and the blood collection fraction between the gas gaps cd is further pushed forward. Further, as shown in FIG. 6B, the pinch valve 3) is closed, the pinch valve 4) of the waste liquid flow path is opened, and a part of the gas gap c is completely discharged. Here, as shown in FIG. 6C, the pinch valve 4 is closed, only the pinch valve 2) to the gas source is opened, the syringe is turned into a pull, the gas is sucked, and the gas gap e that enters the tube 16 is formed.

As shown in FIG. 7A, the pinch valve 2) is closed, the pinch valve 4) of the waste liquid flow path is opened, the syringe is turned into the push state, and the washing liquid mixed with the blood is discharged. . Next, as shown in FIG. 7B,
The pinch valve 4) is closed, the pinch valve 3) is opened, and the blood collection fraction between the gas gaps dc is further advanced in the flow path 17 leading to the blood storage unit. Further, as shown in FIG. 7C, while continuing the push state, the pinch valve
3) is closed, the pinch valve 4) is opened, and the gas gap e and the cleaning liquid before and after the gas gap e and the gas gap a are led to the waste liquid flow path.

Next, as shown in FIG. 8A, in a further pushed state, the pinch valve 1) is opened,
The pinch valve 4) is closed, and the blood fraction below the gas gap b is returned to the animal body. From this state, as shown in FIG. 8B, the syringe is turned into a pull, and the pinch valve 1) is closed and the pinch valve 2) is opened.
Thereby, a gas gap f is formed in the joint opening to the syringe flow path 16. Further, as shown in FIG. 8C, the pinch valve 2) is closed, the pinch valve 1) is opened, and the washing liquid is sent to the animal-side flow path 7 in the same push state of the syringe, so that the collected blood is substantially, that is, the blood mixture. Return all but the cleaning solution to the animal.

As shown in FIG. 9A, the syringe is turned into a pull, and then the pinch valve 1) is closed and the pinch valve 2) is opened. As a result, a gas gap g for the next sampling is formed by the gas introduced from the intake port 14. Next, as shown in FIG. 9B, the syringe is turned to the push state, the pinch valve 2) is closed, and the pinch valve 1) is opened. As a result, the gas gap g moves up to just before the animal 15, and the blood component in front of the gas gap g is completely returned into the animal body 15.
As shown in C, by closing the pinch valve 1) and opening the pinch valve 3), the blood collection fraction between the gas gaps bc is moved in the tube 17 to just before the blood storage unit.

As shown in FIG. 10A, the feeding of the blood collection fraction is further continued, and the sample collection vial 18 in the blood storage unit contains the blood collection fraction between gas gaps cd and up to half of the gas gap c behind it. It is supplied to a sample vial 18. Sample vial 18 contains
An undiluted blood sample will be provided. The tube 17 returns to the washing port, and this state is continued as shown in FIG. 10B, and the washing of the tube 17 is completed. Further, as shown in FIG. 10C, the syringe is in a pull state, in which the pinch valve 3) is closed, and the pinch valve 2) to the intake port 14 is opened to wash the flow path to the blood storage unit. Gas gap h is formed.

FIG. 11 is a view showing a final cleaning step. As shown in FIG. 11A, the syringe is switched to the push state, the pinch valve 2) is closed, the pinch valve 4) is opened, and the gas is removed. The cleaning liquid is discharged from the waste liquid port through the gap h. Further, as shown in FIG. 11B, the push state of the syringe is continued. Here, the pinch valve 3) to the blood storage unit is opened, the pinch valve 4) is closed, and the cleaning liquid behind the gas gap h is supplied to the blood storage unit. Into the tube 17. Further, as shown in FIG. 11C, by closing the pinch valve 3) and opening the pinch valve 4), the remaining part of the gas gap that did not go to the blood storage part in the further push state of the syringe is released. The cleaning liquid is drained until it runs out.

In this state, the pinch valve 4) of the waste liquid passage is closed to return to the standby state as shown in FIG. 3A again.

[0028]

According to the present invention, as described above, the pinch cock is formed by the flexible tube rather than the rotating mechanism of the stopper, so that clogging due to coagulation of residual blood and the like is unlikely to occur, and the liquid is collected by the automatic sampling method. A gas gap was formed before and after the fraction, so that the cleaning liquid was not mixed, and the reproducibility of collection was extremely good. An object of the present invention is to provide a biological fluid automatic sampling device.

Measurement of Reproducibility of Collected Blood A catheter was implanted into the heart (right atrium) of a male Wistar rat weighing 280 g, and after waiting for recovery for two days, 30 μl was collected every 15 minutes 19 times. The measurement method is as follows: 30 μl of 1
A 0 mM tris-HCl buffer (pH 7.4) was added to lyse the cells, and a 50-fold dilution was performed. Add 20 μl of this solution to U
The mixture was injected into a V meter, and the absorbance at 580 mm was measured.
This 20 μl injection / absorbance measurement per sample is 3
This was repeated twice, and the variation in blood collection was calculated based on the average value. As a result, a good result of ± 5% was obtained. Table 1 shows the results.

[0030]

[Table 1]

[Brief description of the drawings]

FIG. 1 is a perspective view showing a configuration example of an animal blood four-channel automatic sampling device incorporating a biological fluid automatic sampling mechanism of the present invention.

FIG. 2 is a perspective view showing a structure and a flow path connection of a five-way joint incorporated in the apparatus of FIG.

FIG. 3 shows all connection flow paths showing the states of A: standby state, B: formation of a cleaning gas gap, and C: start of blood withdrawal when performing automatic sampling of animal blood by the apparatus configuration of the present invention. FIG.

FIG. 4 is a schematic view of a similar total connection channel showing each state of A: formation of a gas gap before sample, B: formation of a blood sample fraction, and C: formation of a gas gap after sample, following the process of FIG. is there.

FIG. 5: Following the process of FIG. 4, A: transfer of the blood sample between the gas gaps to the storage channel, B: extra disposal of the sample trailing gap, and C: return of the preceding blood fraction to the animal channel. 4 is a schematic view of a similar all connection flow path showing the respective states of FIG.

FIG. 6: Following the process of FIG. 5, A: 5-way joint wash # 1 before returning blood to the animal, B: Same wash # 2, and C:
It is the schematic of the same all connection flow path which shows each state of the same washing | cleaning # 3.

FIG. 7: Following the process of FIG. 6, A: 5-way joint wash # 4 before returning blood to the animal, B: Same wash # 5, and C:
It is the schematic of the same whole connection flow path which shows each state of the same washing | cleaning # 6.

FIG. 8: A: blood return to the animal body following the process of FIG. 7
1, B: introduction of gas gap into the animal body flow path and C: blood return to the animal body # 2.

FIG. 9 is a flow chart of the process shown in FIG. 8, wherein A: formation of a gas gap for the next sample, B: return of blood to the animal, and C:
It is a schematic diagram of the same whole connection flow path which shows each state of preparation for introduction of a blood sample into a storage vial.

FIG. 10 is a view showing the states of A: introduction of a blood sample into a vial # 1, B: completion of introduction of a blood sample into a vial, and C: formation of a cleaning gas gap following the step of FIG. It is the schematic of all the connection flow paths.

FIG. 11 is a flow chart illustrating the process of FIG. 10;
1, B: Final cleaning # 2, and C: Final cleaning completion (return to the standby state when the waste liquid flow path cock is closed in this state).

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Operator operation part 2 Push-pull syringe part 3 Blood storage part 4X X-axis drive part 4Y Y-axis drive part 5 Blood collection flow path part 5a-5d Joint box 6a-6d Experimental animal part 7 Spout 8 Five-way joint 8a-8d 4-way port 9a-9d Connection base 10 Solenoid valve 11 Syringe 12 Washing liquid reservoir 13 Pressure gauge 14 Inlet port 15 Animal 16 Tube 17 Blood sampling fraction

Continuation of front page (56) References JP-A-8-178806 (JP, A) JP-A-10-153532 (JP, A) JP-A-7-148185 (JP, A) JP-A-5-11059 (JP, A) , A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 1/00-1/44 G01N 35/00-35/10 A61B 5/15

Claims (3)

(57) [Claims] 1. An apparatus for automatically collecting biological fluid of an animal at an arbitrary interval, wherein the five ports of a five-way joint are connected to five tubes, respectively, and at least four of the connecting bases are connected to the five tubes. The flow path can be closed by its own deformation, and the tip of the first tube connected to the first port is connected to the biological fluid collection means embedded in the animal body, and the second port is connected to the second port. The distal end of the connected second tube is opened as an air inlet, and the distal end of the third tube connected to the third port is connected to the biological fluid sample control-operated fractionating means, and the fourth port is connected to the fourth port. The tip of the connected fourth tube is opened as a waste liquid port, the tip of the fifth tube connected to the neutral port is connected to a control-operable syringe, and a branch flow path to the washing liquid reservoir immediately before the syringe. Contact Subsequently, the connection bases of the first to fourth tubes are selectively bent and deformed according to time-series control related to each control operation of the syringe and the sorting unit, thereby closing the flow path, A sampling fraction from an animal body divided into front and rear by a gas gap sucked and formed from an inlet is formed in a fifth tube, and then supplied to the sorting means via a third tube. A five-way joint including a gas gap formed in a portion inside the first tube immediately before the biological fluid collecting means, four tubes excluding the gas gap and the second tube, and a four-way port corresponding to the inside of the connection base. An automatic sampling apparatus for biological fluid, wherein the inside of the flow path is filled with the washing liquid. 2. The method according to claim 2, wherein two sampling fractions are formed in series in the fifth tube, the first fraction is returned to the animal body, and the second fraction is supplied to the sorting means. The apparatus according to claim 1, characterized in that: 3. The pressure gauge according to claim 1, wherein a pressure gauge is arranged before the branch flow path in the fifth tube.
Or the apparatus according to 2.