U.S. patent application number 10/336790 was filed with the patent office on 2003-06-19 for sheath liquid supplying apparatus, sheath liquid supplying method, and evaluating method of sheath liquid supplying condition.
This patent application is currently assigned to SYSMEX CORPORATION. Invention is credited to Shibata, Masaharu.
Application Number | 20030110826 10/336790 |
Document ID | / |
Family ID | 18738550 |
Filed Date | 2003-06-19 |
United States Patent
Application |
20030110826 |
Kind Code |
A1 |
Shibata, Masaharu |
June 19, 2003 |
Sheath liquid supplying apparatus, sheath liquid supplying method,
and evaluating method of sheath liquid supplying condition
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.
Inventors: |
Shibata, Masaharu;
(Kobe-shi, JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
SYSMEX CORPORATION
|
Family ID: |
18738550 |
Appl. No.: |
10/336790 |
Filed: |
January 6, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10336790 |
Jan 6, 2003 |
|
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09930967 |
Aug 17, 2001 |
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Current U.S.
Class: |
73/1.36 |
Current CPC
Class: |
B01L 2200/0652 20130101;
B01L 2400/0478 20130101; G01N 2015/1409 20130101; G01N 15/1404
20130101; G01P 21/025 20130101; B01L 3/502776 20130101; B01L
3/502784 20130101; B01L 2400/049 20130101; B01L 2200/0636
20130101 |
Class at
Publication: |
73/1.36 |
International
Class: |
G01F 025/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 18, 2000 |
JP |
2000-248689 |
Claims
What is claimed is:
1. A sheath liquid supplying apparatus comprising: 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.
2. A sheath liquid supplying apparatus claimed in claim 1, wherein
the cylinder has a negative pressure introducing hole positioned at
a side wall thereof for introducing a negative pressure into the
cylinder.
3. A sheath liquid supplying apparatus claimed in claim 2, wherein
the negative pressure introducing hole is positioned closer to the
injection/suction hole compared to the gas introducing hole with
respect to an axial direction of the cylinder.
4. The sheath liquid supplying apparatus claimed in claim 1,
wherein the piston or cylinder is glass, vinyl chloride or
stainless steel.
5. The sheath liquid supplying apparatus claimed in claim 1,
wherein the syringe has a capacity of about 3-5 mL.
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 .sctn.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.sup.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] FIG. 1 is a side view showing an embodiment of a sheath
liquid supplying apparatus according to the present invention;
[0012] FIG. 2 is a view in section taken along the line A-A in FIG.
1;
[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] FIG. 4 is an explanatory view showing an operation of a
syringe in the embodiment of the present invention;
[0015] FIG. 5 is an explanatory view showing an operation of a
syringe in the embodiment of the present invention;
[0016] FIG. 6 is an explanatory view showing an operation of a
syringe in the embodiment of the present invention;
[0017] FIG. 7 is an explanatory view showing an operation of a
syringe in the embodiment of the present invention;
[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] 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] FIG. 10 is a waveform view showing a performance of a
comparative example;
[0021] FIG. 11 is a waveform view showing a performance of the
embodiment of the present invention; and
[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] 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 64a, 65a and 66a 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 71a mounted to an output
shaft of the stepping motor 69, a follower pulley 71b rotatably
supported by the frame 68 and an endless belt 72 bridged between
the pulleys 71a and 71b.
[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 71a, 71b 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] 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 5a 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 5a of the sheath
liquid injecting section 5 in the sheath flow cell 1.
[0046] Subsequently, a piston 44b of the metering syringe 44 is
driven by a motor 44a, 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 44b 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] 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 N.sub.T is
prepared as a sheath liquid, while a liquid having a refractive
index N.sub.3 (not equal to N.sub.T) 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 N.sub.T and N.sub.S were prepared as the sheath
liquid and sample liquid.
[0060] N.sub.T=1.341
[0061] N.sub.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
.mu.L/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] 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 waveform 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.
* * * * *