Abstract:
A sheath liquid supplying apparatus includes a syringe including a piston and a cylinder slidably accommodating the piston and a stepping motor for causing the piston to slide in the cylinder, wherein the cylinder has an injection/suction hole of a sheath liquid positioned at a distal end thereof and a gas introducing hole positioned at a side wall thereof for introducing gas into the cylinder.

Description:
CROSS-REFERENCE TO RELATED APPLICATION  
       [0001]    This application is related to Japanese Patent Application No. 2000-248689 filed in Aug. 18, 2000, whose priority is claimed under 35 USC §119, the disclosure of which is incorporated by reference in its entirety. 
     
    
     
       BACKGROUND OF THE INVENTION  
         [0002]    1. Field of the Invention  
           [0003]    The present invention relates to a sheath liquid supplying apparatus, a sheath liquid supplying method and an evaluating method of a sheath liquid supplying condition, and more particularly to an apparatus or method for supplying a sheath liquid to a sheath flow cell by using a syringe in a flow cytometer.  
           [0004]    2. Description of the Related Arts  
           [0005]    A flow cytometer using a so-called sheath flow method has been well-known as an apparatus for analyzing particles such as a cell, blood cell, or the like in a sample. In this method, a sheath liquid is flown around a sample solution (particle-floating solution) ejected from a nozzle in a sheath flow cell to form a sheath flow, whereby the sample solution can be converged into a small flow in the sheath flow cell. The converged sample solution is optically measured for analyzing particles in the sample solution. The “sheath flow” means a flow for causing particles in the particle-floating solution (sample solution) pass therethrough in a line with precision by converging the sample solution into a small flow having an outside diameter approximately same as that of the particle at the center portion of the sheath liquid that is flowing through an orifice in a laminar flow state. Analysis of various cells has been performed by using a sample solution obtained by adjusting a sample of blood or urine with a dyeing solution, hemolytic agent or reaction reagent.  
           [0006]    The sample solution is supplied to the sheath flow cell by a syringe having high quantity accuracy. The sheath liquid is supplied to the sheath flow cell by using a method of applying a predetermined positive pressure (0.2-1.6 kgf/cm 2 ) to the sheath liquid chamber (referring to Japanese Unexamined Publication No. HEI 10(1998)-260129).  
           [0007]    However, the supply of the sheath liquid to the sheath flow cell by using a method of applying the positive pressure to the sheath liquid chamber brings a change in viscosity of the sheath liquid in the case of changing the environmental temperature. This change in viscosity brings a change in flow velocity, thereby giving an adverse influence to the optical measurement.  
           [0008]    Accordingly, this method requires a sheath liquid temperature adjusting function for maintaining a constant flow velocity of the sheath liquid, as well as requires an air compressor for applying the positive pressure to the sheath liquid chamber or a regulator for adjusting a pressure. This causes a problem that a pressure-adjusting and sheath liquid supplying apparatus is made complicated.  
           [0009]    In case where the sheath liquid is supplied to the sheath flow cell by using a syringe that is driven by a stepping motor, the flow velocity is not affected by a change in the environmental temperature. However, the flow velocity of the sheath liquid changes depending upon a mechanical factor such as a pulse of a torque of the stepping motor, whereby a ripple occurs on the sheath liquid and sample solution in the sheath flow cell. Specifically, the sheath flow is brought into an unstable state. As a result, a ground noise of an optical detecting signal is fluctuated, which gives an adverse effect on the optical measurement.  
         SUMMARY OF THE INVENTION  
         [0010]    The present invention is accomplished in view of the above circumstances, and aims to provide a sheath liquid supplying apparatus, a sheath liquid supplying method, a flow cytometer including said apparatus, and an evaluating method of a sheath liquid supplying condition wherein a sheath liquid is supplied by using a syringe that is driven by a driving motor. This construction provides that the flow velocity is not affected by an environmental temperature, and prevents a fluctuation in the flow velocity of the sheath liquid due to a mechanical factor such as a pulse of a torque of a stepping motor as well as prevents a fluctuation in a ground noise of an optical detecting signal brought with the fluctuation in the flow velocity. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0011]    [0011]FIG. 1 is a side view showing an embodiment of a sheath liquid supplying apparatus according to the present invention;  
         [0012]    [0012]FIG. 2 is a view in section taken along the line A-A in FIG. 1;  
         [0013]    [0013]FIG. 3 is a systematic view showing a fluid system of an analyzer of material components in urine that includes the sheath liquid supplying apparatus of the present invention;  
         [0014]    [0014]FIG. 4 is an explanatory view showing an operation of a syringe in the embodiment of the present invention;  
         [0015]    [0015]FIG. 5 is an explanatory view showing an operation of a syringe in the embodiment of the present invention;  
         [0016]    [0016]FIG. 6 is an explanatory view showing an operation of a syringe in the embodiment of the present invention;  
         [0017]    [0017]FIG. 7 is an explanatory view showing an operation of a syringe in the embodiment of the present invention;  
         [0018]    [0018]FIG. 8 is a systematic view showing an optical system of an analyzer of material components in urine that includes the sheath liquid supplying apparatus of the present invention;  
         [0019]    [0019]FIG. 9 is a sectional view showing a sheath flow cell of the analyzer of material components in urine to which the sheath liquid supplying apparatus of the present invention is adapted;  
         [0020]    [0020]FIG. 10 is a waveform view showing a performance of a comparative example;  
         [0021]    [0021]FIG. 11 is a waveform view showing a performance of the embodiment of the present invention; and  
         [0022]    [0022]FIG. 12 is an enlarged waveform view showing a performance of the comparative example. 
     
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT  
       [0023]    The present invention will be explained in detail with reference to the drawings. The present invention is not limited to the following explanation.  
         [0024]    Construction of a Sheath Liquid Supplying Apparatus  
         [0025]    [0025]FIG. 1 is a side view showing an embodiment of a sheath liquid supplying apparatus according to the present invention and FIG. 2 is a view in section taken along the line A-A in FIG. 1.  
         [0026]    As shown in these figures, a main body  60  of a supplying apparatus comprises a syringe  61  and a driving apparatus  70 . The syringe  61  has a piston  62  and a cylinder  63  that accommodates the piston  62  so as to be slidable in a direction shown by arrows B and C. The arrow B expresses up direction along the axial direction of the cylinder  63 , and the arrow C expresses down direction along the axial direction of the cylinder  63 . The piston  62  and cylinder  63  can be made of a material having a chemical resistance, such as glass, vinyl chloride, stainless steel or the like. It is necessary for the syringe  61  to have a capacity, which can at least supply a sheath liquid required for one measurement in a sheath flow cell. For example, the capacity may be approximately 3 to 5 mL.  
         [0027]    The cylinder  63  is provided with an injection/suction hole  64  of the sheath liquid at its bottom edge that injects the sheath liquid to the cylinder  63  and extracts the sheath liquid from the cylinder  63  corresponding to the forward and rearward motions of the piston  62 . Provided also at the cylinder  63  are a gas introducing hole  65  and a negative pressure introducing hole  66  at its side wall. The gas introducing hole  65  introduces a gas (for example, an air) between a liquid surface of the sheath liquid accommodated in the cylinder  63  and the piston  62  to form a gas layer. A buffer action of the gas layer absorbs a rotational irregularity (periodical fluctuation of a torque), to thereby stabilize the ejecting velocity of the sheath liquid. Accordingly, the gas introducing hole  65  is mounted in the vicinity of a piston inserting opening of the cylinder  63 .  
         [0028]    A circular seal member  67  is disposed at the upper inside surface of the cylinder  63  for sealing up the inside surface of the cylinder  63  and the outside surface of the piston  62 .  
         [0029]    The negative pressure introducing hole  66  is provided closer to the injection/suction hole  64  compared to the gas introducing hole  65  with respect to the axial direction of the cylinder  63 . Nipples  64   a ,  65   a  and  66   a  for connecting an external tube are respectively disposed at the injection/suction hole  64 , gas introducing hole  65  and negative pressure introducing hole  66  of the syringe  61 .  
         [0030]    A driving apparatus  70  has a frame  68 , a stepping motor  69  mounted to the frame  68 , a driving pulley  71   a  mounted to an output shaft of the stepping motor  69 , a follower pulley  71   b  rotatably supported by the frame  68  and an endless belt  72  bridged between the pulleys  71   a  and  71   b.    
         [0031]    The frame  68  has a slide shaft  73  mounted along the axial direction of the cylinder  63  that supports a sliding member  74  so as to be capable of sliding in the direction shown by arrows B and C. The sliding member  74  has arms  75  and  76  which horizontally project to respectively connect to the upper edge of the piston  62  and the endless belt  72 .  
         [0032]    When the stepping motor  69  rotates, its rotational motion is converted into the linear motion by the pulley  71   a ,  71   b  and the endless belt  72 . The linear motion is transmitted to the piston  62  via the arm  76 , sliding member  74  and arm  75 , whereby the piston  62  can be driven in the directions of B and C.  
         [0033]    A motor now on sale can be used as the stepping motor  69 . For example, the stepping motor of PK43 AG470-100(12)6TA-3 manufactured by Sanryu Co., Ltd. can be used as the stepping motor  69  causing the piston  62  to slide in the cylinder  63 .  
         [0034]    Analyzer of Material Components in Urine to which Sheath Liquid Supplying Apparatus is adapted  
         [0035]    [0035]FIG. 3 is a systematic view showing a flow system where the sheath liquid supplying apparatus in FIG. 1 is adapted to an analyzer of material components in urine. The analyzer is a so-called flow cytometer. Each component of the flow system is connected by flow path of a tube network TN.  
         [0036]    A sheath flow cell  1  in this analyzer of material components in urine has a construction shown in FIG. 9. A sheath liquid is supplied from the sheath liquid supplying apparatus  60  to an injection hole  5   a  of a sheath liquid injecting section  5 . A sample liquid is supplied to a nozzle  6  from a syringe  44  for supplying a sample liquid.  
         [0037]    In an initial condition, the sheath liquid supplying apparatus  60  is in a condition where the leading edge of the piston  62  nearly reaches the injection/suction hole  64  at the bottom of the cylinder  63  as shown in FIG. 4. When a valve  50  is opened to lift the piston  62  in the direction of B (see FIG. 1), the sheath liquid in an open-air sheath liquid chamber  42  is sucked in the cylinder  63  via a valve  50 . When the leading edge of the piston  62  reaches the vicinity of the negative pressure introducing hole  66  as shown in FIG. 5, the piston  62  temporarily stops rising, and then, a valve  51  is opened.  
         [0038]    By this operation, a negative pressure of a suction apparatus  49  is applied to the inside of the cylinder  63  via the negative pressure introducing hole  66 , whereby the sheath liquid is sucked into the suction apparatus  49  via the chamber  42 , valve  50 , injection/suction hole  64 , negative pressure introducing hole  66  and valve  51 , so that bubbles are eliminated from the sheath liquid sucked into the cylinder  63 .  
         [0039]    Subsequently, the valve  51  is closed and the valve  52  is opened, with the result that the piston  62  moves upward until its leading edge passes a little through the gas introducing hole  65 . By this operation, air is introduced from the gas introducing hole  65  to thereby form an air layer G (hereinafter referred to as air damper) between the sheath liquid surface and the leading edge of the piston  62 .  
         [0040]    After the air damper having a predetermined volume is formed, the piston  62  terminates, and the valves  50  and  52  are closed. The sheath liquid supplying apparatus  60  finishes here the preparation for supplying the sheath liquid. A washing process and measuring process are executed as follows.  
         [0041]    Washing process  
         [0042]    Firstly, valves  41 ,  47  and  50  are opened for sucking the sheath liquid with the negative pressure of the suction apparatus  49  from the open-air sheath chamber  42  accommodating the sheath liquid. The sheath liquid is discharged to the suction apparatus  49  via the valve  50 , sheath flow cell  1 , nozzle  6  and valve  47 , and at the same time, discharged to the suction apparatus  49  via a metering syringe  44  for supplying a sample liquid and the valve  47 . Thereafter, the valves  41 ,  47  and  50  are closed after a predetermined period. By this operation, the metering syringe  44 , nozzle  6 , sheath flow cell  1  and its flow path are washed with the sheath liquid.  
         [0043]    Measuring process  
         [0044]    Subsequently, valves  46  and  47  are opened for sucking a sample liquid with the negative pressure of the suction apparatus  49  from a reaction chamber  48  in which a sample liquid containing material components in urine is reacted with a reactant and the resultant is accommodated. When the sheath liquid in the flow path between the valve  46  and the nozzle  6  is replaced with the sample liquid, the valves  46  and  47  are closed.  
         [0045]    Next, the valve  53  is opened and the stepping motor  69  of the sheath liquid supplying apparatus  60  is driven for moving the piston  62  toward the injection/suction hole  64  as shown in FIG. 7. By this operation, the sheath liquid in the cylinder  63  is supplied to the sheath flow cell  1  via the injection/suction hole  64 , whereby it is injected to the injection hole  5   a  of the sheath liquid injecting section  5  in the sheath flow cell l.  
         [0046]    Subsequently, a piston  44   b  of the metering syringe  44  is driven by a motor  44   a , whereby the sample liquid present between the valve  46  and the nozzle  6  is ejected from the nozzle  6  as shown in FIG. 9. The ejected sample liquid is converged into a small flow with the sheath liquid for passing through an orifice  13 , and then, discharged to an open-air discharge liquid chamber  45  with the sheath liquid.  
         [0047]    A laser beam L is irradiated to the orifice portion  13  as described later for optically measuring material components in urine among the sample liquid. Thereafter, the piston  44   b  of the metering syringe  44  is driven during a predetermined period to supply the predetermined amount of sample liquid to the sheath flow cell  1 . Then, the valve  53  is closed to finish the measuring process. The valve  57  is opened, as necessity requires, for discharging the sheath liquid and sample liquid accommodated in the discharge liquid chamber  45 .  
         [0048]    In the above measuring process, the sheath liquid is pushed by the piston  62  driven by the stepping motor  69  to be supplied from the cylinder  63  to the sheath flow cell  1 . The stepping motor originally has a rotational irregularity (periodical fluctuation of torque). This rotational irregularity is absorbed by a buffer operation of the air damper G shown in FIG. 7. As a result, the sheath liquid is smoothly supplied to the sheath flow cell  1  with a constant flow velocity without generating a fluctuation in flow velocity as shown in the result of a performance test described later.  
         [0049]    When the measuring process is finished in this way, the process for injecting the sheath liquid to the cylinder  63  of the sheath liquid supplying apparatus  60  and the washing process are performed to make preparations for the next process.  
         [0050]    [0050]FIG. 8 is a perspective view showing an optical system of the analyzer of material components in urine. In the same figure, a laser beam L emerged from a laser diode  21  irradiates the orifice portion  13  of the sheath flow cell  1  via a collimator lens  22 . The forward scattered light emerging from the material components in urine which pass through the orifice portion  13  is incident to a photodiode  26  via a focusing lens  24  and a pinhole plate  25 .  
         [0051]    On the other hand, the sideward scattered light emerging from the material components in urine which pass through the orifice portion  13  is incident to a photomultiplier tube (hereinafter referred to as photomul) via a focusing lens  27  and a dichroic mirror  28 , while the sideward fluorescence emerging from the material components in urine which pass through the orifice portion  13  is incident to a photomul  31  via the focusing lens  27 , dichoric mirror  28 , a filter  36  and a pinhole plate  30 .  
         [0052]    The forward scattered light signal outputted from the photodiode  26 , the sideward scattered light signal outputted from the photomul  29  and the sideward fluorescence signal outputted from the photomul  31  are respectively amplified by each amplifier  32 ,  33  and  34 , and then, inputted to an analyzing section  35 . The analyzing section  35  is comprised of a microcomputer that processes and analyzes the output signals from the photodiode  26 , photomul  29  and  31  based upon a predetermined program and outputs the resultant to a display device or a printer.  
         [0053]    Performance Test of Sheath Liquid Supplying Apparatus  
         [0054]    A fluctuation in flow velocity (irregularity in flow velocity) of the sheath liquid in the sheath liquid supplying apparatus  60  can be examined by the following manner by using the analyzer of material components in urine shown in FIGS. 3 and 8.  
         [0055]    Firstly, a liquid having a refractive index NT is prepared as a sheath liquid, while a liquid having a refractive index Ns (not equal to NT) is prepared as a sample liquid.  
         [0056]    Subsequently, prepared each liquid is supplied to the sheath flow cell  1  by using the flow system shown in FIG. 3. The laser beam L is irradiated to the orifice portion  13  of the sheath flow cell  1  with the optical system of FIG. 8. The photodiode  26  detects its scattered light intensity and the detected light is amplified by the amplifier  32 .  
         [0057]    The amplified signal waveform is observed by an oscilloscope.  
         [0058]    When the ripple occurs in the sample liquid flow at the orifice portion  13  due to the fluctuation in flow velocity of the sheath liquid, the scattered light intensity from the laser beam L changes because of the difference in refractive index between the sheath liquid and the sample liquid flow, whereby the output signal from the amplifier  32  fluctuates (it is considered that the fluctuation in the output signal depends upon “each refractive index of sample liquid and sheath liquid” and “a width of ripple in sample liquid flow”).  
         [0059]    In view of this, liquids each having the following refractive indices NT and Ns were prepared as the sheath liquid and sample liquid.  
         [0060]    N T =1.341  
         [0061]    N S =1.334  
         [0062]    Note that liquids each having a different refractive index can be adjusted, for example, with solutions of salt each having a different concentration.  
         [0063]    The flow amount of the sample liquid was set to 1.7 μL/seconds, while the flow velocity of the sample liquid was set to 7.5 m/seconds that can obtain a laminar flow. The output waveform from the amplifier  32  was recorded with the oscilloscope with respect to the presence of the air damper G (FIGS. 6 and 7). The results are shown in FIGS. 10 and 11. FIG. 10 represents the case where the air damper G is not formed, while FIG. 11 represents the case where the air damper G is formed.  
         [0064]    In FIG. 12, a waveform (a) is obtained by recording the waveform in FIG. 10 with a tenfold time axis, while a waveform (b) represents a waveform of a driving pulse of the stepping motor  69  (FIG. 1) corresponding to the waveform (a).  
         [0065]    It is understood from FIG. 10 that the waveform has a large fluctuation (ripple) to cause a great ripple in the sample liquid flow at the orifice portion  13  in case where the air damper G is not formed. On the other hand, it is understood from FIG. 11 that the waveform has a small fluctuation to thereby prevent the ripple from occurring in the sample liquid flow in case where the air damper G is formed. Consequently, the formation of the air damper G brings a stable flow of the sample liquid.  
         [0066]    [0066]FIG. 12 represents that a main cause of the fluctuation in the waveform (a) is caused by the rotational irregularity (periodical fluctuation in torque) of the stepping motor  69  since the correlation is periodically established between the waveform (a) and the waveform (b). FIG. 10 represents that the rotational irregularity is effectively absorbed by the air damper G.  
         [0067]    It is understood that the minimum volume necessary for the air damper G may be set to the one that brings a minimum amplitude of the wave form of FIG. 10.  
         [0068]    According to a sheath liquid supplying apparatus and its method of the present invention, the supplying apparatus itself can simply be realized with a combination of a syringe and a stepping motor. Further, gas is intervened between a leading edge of a piston in the syringe and a sheath liquid in the cylinder for absorbing a fluctuation of the piston due to the stepping motor, whereby the sheath liquid can be supplied to a sheath flow cell with a constant stable speed.  
         [0069]    Moreover, according to an evaluating method of a sheath liquid supplying condition, liquids each having a different refractive index are supplied to a sheath flow cell as a sheath liquid and a sample liquid, and a degree of the fluctuation in the scattered light intensity upon irradiating light to the sheath flow cell teaches information such as a periodical change, fluctuation period, fluctuation width or the like can be obtained with respect to the supplying condition of the sheath liquid. Specifically, it is possible to simplify the evaluating method.