JPH08170960A - Reagent for analyzing corpuscle component in urine and method for analyzing corpuscle component using reagent - Google Patents

Abstract

PURPOSE: To obtain a reagent for accurately analyzing a corpuscle component in urine by containing a buffer agent for maintaining pH within a specific range, an osmotic pressure compensating agent for maintaining an osmotic pressure within a specific range, a first dye of condensation benzene derivative, and a second fluorescent dye and chelate agent for dying a damaged cell. CONSTITUTION: An analysis reagent is constituted by containing (1) A buffer agent for maintaining pH at 5.0-9.0, (2) An osmotic pressure compensating agent for maintaining osmotic pressure at 100mOsm/kg-600mOSm/kg, (3) A first dye in condensation benzene system, (4) A second fluorescent dye for dying a damaged cell, and (5) Chelate agent. A corpuscle component in urine to be measured includes especially red cell, white cell, and epithelial cell. A buffer agent is used to constantly maintain the pH of a measurement sample to obtain a stable fluorescent intensity. An osmotic pressure compensating agent is added to prevent the hemolyzation of red blood and to obtain a stable fluorescent intensity.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a reagent for analyzing formed elements in urine and a method for analyzing formed elements using the reagent,
More specifically, the present invention relates to a reagent used for an optical analysis method of formed matter in urine by applying flow cytometry, and an analysis method thereof.

[0002]

2. Description of the Related Art With respect to diseases such as renal / urinary tract infections, inflammatory lesions, degenerative lesions, stone diseases, and tumors, various formed components appear in urine according to the respective diseases. Examples of the material include erythrocytes, leukocytes, epithelial cells, casts, bacteria, fungi, crystals, mucus threads and the like. Analyzing these components in urine is especially important for early detection of renal and urinary tract diseases and estimation of abnormal sites. For example, the measurement of red blood cells is important in determining the presence or absence of bleeding in the pathway from the glomerulus of the kidney to the urethra, and the appearance of white blood cells is
Suspected renal diseases such as pyelonephritis are suspected, and inflammation and infection can be detected early. In addition, by examining the morphology of casts and red blood cells, the site of origin can be estimated.

[0003] Conventionally, visual analysis has been widely performed with a microscope for the analysis of formed components in urine. First, urine is concentrated by centrifugation, and the precipitate is optionally stained, loaded on a microscope slide, and classified and counted under a microscope. Further, in recent years, an automatic measuring device combining a flat sheath flow and an image processing technique has been developed. This is a urine sample solution with a sheath liquid as the outer layer, adjusted to an extremely flat flow, is photographed with a video camera, and this static screen is image-processed to cut out and display the image of the formed component in the sample solution. To do. The technician looks at the display to determine the formed portion, and the classification process is performed.

Further, Japanese Patent Laid-Open No. 4-337459 discloses a method for automatically classifying and counting formed elements in urine.
A reagent for analyzing cells in urine to which flow cytometry is applied and a method thereof are disclosed. The reagents include fluorescent dyes, osmotic compensators and buffers, and various fluorescent dyes,
An osmotic pressure compensating agent and a buffering agent are disclosed, and a reagent using Neutral Red or Auramine O as a fluorescent dye is described in Examples.

[0005]

By the way, it is desirable to test urine samples as soon as possible after collection, since changes such as denaturation of formed components and increase in the number of bacteria occur over time after collection. . In the visual inspection with a microscope, pretreatment such as centrifugation and concentration of a urine sample takes time and labor, and the microscopic work puts a heavy burden on the technician.
Further, the inspection accuracy is low because the number of observed cells is small.

On the other hand, in the automatic measuring device using the image processing technique, although it is advantageous in that the burden is reduced as compared with the microscopic inspection work, the inspection technician must determine the formed portion, Since the processing speed is slow, it is not always satisfactory when the number of samples is large. Further, in any of the methods of the visual inspection and the automatic measuring device by the image processing, it is necessary to have a skill to discriminate the formed matter.

The method in which the flow cytometry of Japanese Patent Laid-Open No. 4-337459 is applied to urine analysis is advantageous in that it allows rapid measurement, but as a result of further studies,
It turned out that there were the following problems.

(1) When crystals appear in urine, it is difficult to distinguish them from red blood cells. (2) It is difficult to classify other cellular components in a sample in which many amorphous salts have appeared. (3) Shown in the embodiment of JP-A-4-337459
When an urine O-containing reagent having a pH of 8.5 is used to measure a urine sample containing hemoglobin or protein, the hemoglobin or protein may be bound to the dye to form a microprecipitate, which prevents measurement. (4) (3) can be solved by making the pH acidic, but the stainability becomes poor, and when yeast-like fungi appear, it is difficult to distinguish from red blood cells.

(5) When analyzing urine with a flow cytometer, the dilution ratio must be kept low because the amount of formed elements present in the urine is small. When fluorescent substances such as drugs and antibiotics are excreted, the fluorescence behind the urine itself (background noise) cannot be ignored, and the fluorescence signal intensity of tangible particles may not be obtained. is there.
Further, when Neutral Red is used, the influence of back fluorescence due to the dye that is not bound to cells is large.

The present invention has been made in view of the above problems, and it is an object of the present invention to more accurately analyze formed elements in urine.

[0011]

According to the present invention, (i) pH
Buffering agent to maintain the value of 5.0 to 9.0, (ii) osmotic pressure of 100 mOsm /
An osmotic pressure compensating agent for maintaining Kg to 600 mOsm / Kg, (iii) a first dye of a condensed benzene derivative system, (iv) a second fluorescent dye capable of staining damaged cells, and (v) a chelating agent. There is provided a reagent for analyzing formed particles in urine, which comprises:

According to the present invention from another point of view, (i)
Buffering agent to keep pH at 5.0-9.0, (ii) Osmotic pressure 100mOs
There is provided a reagent for analyzing formed elements in urine, which comprises an osmotic pressure compensating agent for maintaining m / Kg to 600 mOsm / Kg, (iii) a dye excitable at a red wavelength, and (iv) a chelating agent. It

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, the urinary particles to be measured include erythrocytes, leukocytes, epithelial cells, casts, bacteria, yeast-like fungi and the like. The buffer in the analytical reagent of the present invention is used to keep the pH of the measurement sample within a certain range so that stable fluorescence intensity can be obtained. The pH is adjusted in the range of pH 5.0 to 9.0 to suppress hemolysis of red blood cells. Of the crystalline components in urine, especially amorphous salts can be dissolved by diluting with various aqueous solutions such as physiological saline, dilute hydrochloric acid, dilute acetic acid, and potassium hydroxide aqueous solution. However, some crystalline components in urine are acidic and alkaline. Therefore, at around neutral, pH 6.5 to 7.5 is preferable, and pH 6.8 to 7.2 is more preferable, since amorphous salts that precipitate in acidic and alkaline can be dissolved more rapidly. As the buffering agent, a conventionally known one can be used. For example, Tris and MES, Bis-Tris, ADA, PIPE
S, ACES, MOPSO, BES, MOPS, TES, HEPES, DIPSO, TAPS
Good buffer agents such as O, POPSO, HEPPSO, EPPS, Tricine, Bicine and TAPS can be mentioned. Above all, HE
PES is preferred. The concentration is used at a concentration such that when the urine sample is diluted, the pH is within a certain range, depending on the buffering capacity of the buffer used. Usually 20-500 mM, preferably 50
~ 200 mM.

The osmotic pressure compensating agent is added for the purpose of preventing hemolysis of red blood cells and for obtaining stable fluorescence intensity. The osmotic pressure of urine is
It is distributed over a wide range from 50 to 1300 mOsm / kg. If the osmotic pressure of the analytical reagent is too low, hemolysis of erythrocytes will proceed early, while if it is too high, cell damage will be large. Therefore, the osmotic pressure is preferably 100-600 mOsm / kg, 150-500 mOsm / kg.
More preferred than mOsm / kg. As the osmotic pressure compensating agent, inorganic salts, organic salts such as propionate, saccharides and the like are used. As the inorganic salts, sodium chloride, potassium chloride, lithium chloride and the like, as the propionate, sodium propionate, potassium propionate, ammonium propionate and the like, oxalate as the other organic salts,
Acetate and other sugars include sorbitol, glucose,
Examples include mannitol.

As the first dye, a condensed benzene derivative as shown below can be used.

[0016]

[Chemical 9]

[Wherein A is -O-, -S- or -C (C
H _{3) 2} -, R is a lower alkyl group, X is halogen, Y is -CH = or -NH-, n is 0 or 1, B is

[0018]

[Chemical 10]

(Wherein A and R are as defined above) or two lower alkoxy groups or one di-lower alkyl
Kiramino group (this lower alkyl is substituted with a cyano group
May be present). ]. Up
As the lower alkyl group, an alkyl group having 1 to 6 carbon atoms
Means, for example, methyl, ethyl, propyl, butyl,
Examples include isobutyl, pentyl, hexyl and the like. X's
Examples of halogen atoms include fluorine, chlorine, bromine, and iodine.
You can Also, with two lower alkoxy groups in B
Substituted phenyl group means two C _1-3Alkoxy
A group, preferably C_1-2An alkoxy group such as methoxy
Group, a phenyl group substituted with an ethoxy group. concrete
Include 2,6-dimethoxyphenyl group and 2,6-dieto
A phenyl group is mentioned. Also, the lower grade in B
Alkylamino group (wherein the lower alkyl group is substituted with a cyano group)
The phenyl group substituted with (optionally) is C
_1-3Alkylamino group, preferably C_1-2Alkylami
A phenyl group substituted with a phenyl group. Where the archi
Group may be substituted with a cyano group, for example methyl.
And ethyl, cyanomethyl, cyanoethyl and the like. Good
A di-lower alkylamino group (wherein the lower alkyl is a
Phenyl group substituted with (optionally substituted with
As a 4-dimethylaminophenyl group, 4-diethyl
Luaminophenyl group, 4- (cyanoethylmethylamido)
No) phenyl group and the like.

Specific examples of such a condensed benzene derivative include:

[0021]

[Chemical 11]

[0022]

[Chemical 12]

And the like. Among the above condensed benzene derivatives, the NK-series can be obtained from Japan Photosensitive Dye Research Institute Co., Ltd. Among them, DiOCn (3) (n = 1 to 6), which is an oxacarbocyanine dye, is preferable, and DiOC6
(3) is more preferable. The first dye is not limited to these, and the pH is 5.0 to 9.0, preferably,
Optimum staining pH of 6.5 to 7.5 that binds to cell membranes,
Any component that does not form a precipitate upon binding with a component in urine, such as a protein, can be used. The concentration of the first dye is used in the range where the final concentration (concentration in the measurement sample) is 1 to 30 ppm, preferably 5 to 20 ppm.

The second fluorescent dye is a dye capable of staining damaged cells, for example, EB (ethidium bromide).
Alternatively, PI (propidium iodide) is used. EB is preferably used. The final concentration of the second fluorescent dye is used in the range of 1 to 100 ppm, preferably 30 to 60 ppm.

When the first and second fluorescent dyes are used in combination, light having a blue wavelength can be used as the excitation light source.

Further, in the present invention, instead of using the first and second fluorescent dyes in combination, a dye that can be excited at a red wavelength can be used. As the usable dye, one kind or two kinds out of the group consisting of the following compounds can be used.
Combinations of more than one dye can be used. In this case, the final concentration of the dye that can be excited at the red wavelength is 1 to 300 ppm, and 5 to 100 ppm is preferable.

[0027]

[Chemical 13]

[0028]

Embedded image

[0029]

[Chemical 15]

[0030]

Embedded image

[0031]

[Chemical 17]

[0032]

Embedded image

Among the above dyes, NK-series can be obtained from Japan Photosensitive Dye Research Institute,
Oxazine 4, Oxazine 750 perchlorate, Oxazine 720 is
From Exciton, Inc., Capri Blue GON from Tokyo Kasei Kogyo Co., Ltd., from Basic Green, Iodine Green E. Merck Da
rmstadt, Nile Blue Chloride from Nacalai Tesque, Rhodnile Blue from Aldrich Chemical Compa
ny Inc., from Capri BlueBB is Chroma Gesellshaft Sc
Available from hmid & Co. respectively.

The chelating agent is used to dissolve amorphous salts (for example, ammonium magnesium phosphate, calcium carbonate) appearing in urine. There is no particular limitation as long as it is a calcium-free and magnesium-free agent. For example, EDTA salt, CyDTA, DHEG, DPTA-OH, EDDA, EDDP, GEDT
A, HDTA, HIDA, Methyl-EDTA, NTA, NTP, NTPO, EDDPO
Etc. Preferably, EDTA salt, CyDTA, GEDTA is used. The concentration can be used in the range of 0.05 to 5 W / W%, and preferably 0.1 to 1 W / W%.
It means a compound that is combined with calcium and magnesium ions to form a water-soluble compound.

The reagent of the present invention comprises a buffer, an osmotic pressure compensating agent,
The dye and the chelating agent may be in a one-liquid configuration, but may be in a two-liquid form including a dyeing liquid containing a dye and a diluting liquid containing a buffer, an osmotic pressure compensating agent and a chelating agent. In the case of using a two-component composition of a dyeing solution and a diluting solution, the dye is often unstable in an aqueous solution. Therefore, by dissolving the dye as a dyeing solution in the dye and a water-soluble organic solvent, storage stability can be improved. it can. Furthermore, stabilizers for these dyes may be added to the dyeing solution. In this case, the water-soluble organic solvent that can be used is preferably a lower alkanol, a lower alkylene glycol or a lower alkylene glycol mono-lower alkyl ether. For example, methanol, ethanol, n-propanol, ethylene glycol, diethylene glycol, triethylene glycol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, etc. can be used. Among them, ethylene glycol, diethylene glycol and triethylene glycol are preferable, and ethylene glycol is most preferable in consideration of the influence on cells in urine and viscosity.
Further, an antibacterial agent may be added to the diluted solution in order to prevent bacterial growth during long-term storage. The type of antibacterial agent used is not particularly limited, and triazine antibacterial agents, thiazole antibacterial agents such as BIT (benzisothiazolone), and PTO.
Although a pyridine antibacterial agent such as (pyrithione) can be used, it is preferable to add it at a concentration that does not adversely affect the measurement system. The stabilizer and the antibacterial agent may be added to the one-component reagent.

Further, the electric conductivity of the reagent of the present invention is 1 to 10
When adjusted to mS / cm, preferably 4 to 7 mS / cm,
It is convenient to detect the cylinder by measuring the electrical resistance signal. In the case of buffering agents and osmotic pressure compensating agents with a large ionization degree, adjusting the electric conductivity within the above range will lower the osmotic pressure, which may cause hemolysis of red blood cells in urine. It is preferable to use an organic salt or a non-electrolyte (for example, a saccharide) as the osmotic pressure compensating agent, or to combine them appropriately, because the osmotic pressure can be increased while suppressing the increase in electrical conductivity.

When analyzing formed elements in urine using the reagent of the present invention, raw urine is mixed with the reagent of the present invention. In the one-component reagent containing components (i) to (v) or components (I) to (IV), by mixing with raw urine,
It is diluted 2 to 20 times, and the formed components contained in urine are stained. The dilution ratio of the raw urine is preferably about 2 to 16 times, particularly about 4 to 10 times. At this time, in order to dissolve the amorphous salts contained in urine in a short time, it is preferable to preheat the above reagent at 30 to 40 ° C, preferably 33 to 37 ° C. Mixing with raw urine, in the range of room temperature to 40 ℃, preferably 33 ~ 37 ℃,
It is preferable to carry out for 5 to 60 seconds, preferably 10 to 30 seconds. When the reagent of the present invention is a two-component reagent consisting of a staining liquid and a diluting liquid, the dyeing liquid and the diluting liquid are mixed in advance and then raw urine is added, or the urine and the diluting liquid are mixed. Add post stain. In the case of a two-liquid configuration, it is preferable that the final dilution ratio of urine is 4 to 10 times. In addition, in order to dissolve amorphous salts in a short time, dilute the solution with 30-40
It is preferable to heat at 0 ° C, preferably 33 to 37 ° C.

The urine mixed with the above reagent is made to flow into the flow cell as a urine sample. The urine sample flown in the flow cell is irradiated with excitation light, and the forward scattered light and fluorescence intensity from the formed components in the urine sample are measured. As a result, it is possible to analyze the formed components contained in urine. Further, when measuring the columnar shape, it is convenient to measure the electric resistance signal intensity (volume information) of the urine sample because the detection sensitivity is increased. Therefore, in the urine analysis method of the present invention, it is preferable to use a flow cytometer capable of measuring an electric resistance signal and optical information. The flow cytometer preferably used in the present invention includes the device shown in FIG.

First, by opening the valves 1 and 2 for a predetermined time, the negative pressure from the waste liquid chamber fills the valves 1 and 2 with the sample liquid from the suction nozzle 3. When the syringe 4 pushes out the liquid at a constant flow rate, the sample liquid is discharged from the sample nozzle 6, and at the same time, the valve 8 is opened to supply the sheath liquid to the chamber 7 of the flow cell 5. As a result, the sample is narrowed down in accordance with the inner diameter of the chamber 7 to form a sheath flow, and passes through the orifice 11, as shown in FIG. The shape of the orifice 11 is a prism shape having an inner diameter of 100 to 300 μm on each side, and the material thereof is made of optical glass (including quartz glass). By forming the sheath flow in this way, particles can be made to flow one by one with the centers of the orifices 11 aligned in a line.
The sample liquid and the sheath liquid that have passed through the orifice 11 are discharged through the recovery pipe 14 provided in the chamber 25.

The electrode 13 is made of platinum provided inside the chamber 25 and is a positive electrode, and the electrode 12 is the chamber 7.
The negative electrode is made of stainless steel inside. The electrical resistance between the electrodes 12 and 13 is the resistivity (electrical conductivity) of the sheath liquid, the hole size (hole cross-sectional area) and hole length of the orifice, the resistivity of the sample liquid, and the diameter when the sample liquid is flowing. Depends on

The power source 15 is a DC constant current source, and the electrodes 12 and 1 are
We are supplying between 3 By passing a constant current between the electrodes 12 and 13, a DC voltage determined by the electric resistance and current value between the electrodes 12 and 13 is generated. Particle is orifice 1
After passing 1, the electrical resistance at both ends of the orifice 11 changes. Therefore, the voltage generated between the electrodes 12 and 13 increases only while the particles pass through the orifice 11. Moreover, since a pulsed voltage is generated in proportion to the size of the particles passing through the orifice 11, only this voltage is increased, and this voltage is superimposed on the DC voltage and appears between the electrodes 12 and 13. . Therefore, by detecting this with the amplifier 16, the resistance signal 29 is output.

Sample flow 2 approximately in the center of the orifice 11
The laser light oscillated from the laser 17 is radiated to the laser beam 6 after being focused into an elliptical shape by the condenser lens 18. The shape of the laser light is approximately the same as the blood cell particle diameter in the flow direction of the sample, for example, 1
The shape is narrow around 0 μm, and the shape in the direction orthogonal to the sample flow direction and the irradiation optical axis direction is sufficiently wider than the blood cell particle diameter, for example, about 150 to 300 μm. The laser light applied to the sample flow 26 does not hit the cells (tangible objects) and directly passes through the flow cell 5 and is blocked by the beam stopper 19. The forward scattered light and the forward fluorescence emitted from the cells (tangible objects) at a narrow angle are collected by the collector lens 2
The light is condensed by 0 and passes through the pinhole 21 of the light shielding plate 30. Then, it reaches the dichroic mirror 22.
The fluorescence having a wavelength longer than that of the scattered light passes through the dichroic mirror 22 at a high rate as it is, and after the scattered light is further filtered by the filter 23, the photomultiplier tube (PM
T) 24, the electric signal 27 is detected and output. The scattered light is reflected by the dichroic mirror 22, received by the photodiode 31, converted into an electric signal 28, and output.

According to the reagent for analyzing formed elements in urine of the present invention, the amorphous salt is dissolved by mixing the reagent with urine, and the formed elements in urine are stained so as to be suitable for classification. Will be done.

Amorphous salts that are commonly observed in urine sediments are mostly phosphates and urates, and most of the phosphates are calcium salts. Creates and dissolves a chelating compound of sex. Also, the urate salt is dissolved by dilution and heating. Since these amorphous salts are also found in the urine of healthy persons, their clinical significance is low. Therefore, in order to accurately detect other clinically meaningful formed components (red blood cells, white blood cells, bacteria, yeast-like fungi, casts, etc.), the amorphous salts must be dissolved. However, large crystals, calcium oxalate, and the like have a slow dissolution rate even when a chelating agent is added or a heating treatment, and they are not completely dissolved by the flow cytometer. In addition, pathological crystals such as cystine, leucine, tyrosine, cholesterin, and 2,8DHA are difficult to dissolve even by these operations. For this reason,
The remaining crystals may overlap with the red blood cell region, making it impossible to measure red blood cells accurately. Therefore, in order to prevent these crystals from affecting the measurement of other formed components, it is necessary to strongly stain erythrocytes in particular.

By the way, the fluorescence intensity of the formed components in urine varies from weak to strong depending on the formed components. For example, red blood cells are much weaker than the fluorescence intensity of white blood cells. When white blood cells in blood are measured, white blood cells are strongly stained, and therefore red blood cells that show very weak fluorescence intensity and back fluorescence need not be considered so much. However, when analyzing formed matter in urine, it is necessary to detect even weak fluorescent intensity such as red blood cells, and for that purpose, the detector (PMT) sensitivity is higher than that in the case of measuring white blood cells in blood. Need to raise.
However, if the detector sensitivity is increased, the fluorescence signal intensity of red blood cells may not be obtained and measurement may not be possible due to the influence of the fluorescence behind the urine itself and the fluorescence behind the fluorescent dye that is not bound to cells.

As a result of repeated studies, the present inventor has conducted a fluorescent dye such as DiOCn (3) (n = 1 to
It was found that the problem can be solved by using 6).
The first dye, especially DiOCn (3) (n = 1 to 6), binds to all cell membranes, nuclei and granules by ionic bonding. On the other hand, auramine O as shown in the example of JP-A-4-337459 binds to RNA in cells. Therefore, auramine O hardly stains the cell membrane of erythrocytes, and has the same fluorescence intensity as that of other formed components such as crystals and yeast-like fungi, when these formed components are present in the same sample. Discrimination was difficult. However, DiOCn (3)
By using (n = 1 to 6), the dye binds to and adsorbs on all cell membranes, simultaneously improving the staining properties of red blood cells and yeast-like fungi, and due to the difference in fluorescence intensity, crystals, red blood cells, yeast-like The fungus can be discriminated. On the other hand, white blood cells are stained much more intensely than red blood cells. Further, since the dyeability is improved and the fluorescence intensity is also increased, the influence of the back fluorescence is reduced.

As the second fluorescent dye, one known as a dye for dyeing damaged cells among cells is used. The first dye can stain leukocytes, but the staining of damaged leukocytes is poorer than that of living leukocytes, and is similar to the fluorescence signal intensity of clustered bacteria. When they were present in the same sample, discrimination was difficult. As a dye that compensates for this drawback, it has been found that a dye that stains damaged cells, such as EB, can be used. By using the second fluorescent dye, the damaged white blood cells were stained more strongly than the bacteria, and the bacteria could be distinguished.

The present inventors have also found that the same kind of dye as that described above can be obtained with one type of dye that can be used in the present invention and can be excited at a red wavelength.

Further, by setting the electric conductivity of the reagent of the present invention within the above range, it becomes easy to detect a cylinder. That is, in order to detect the cylinder accurately, it is preferable to measure the forward scattered light and the electric resistance signal. Japanese Patent Laid-Open No. Hei 4
In Japanese Patent No. 337459, a cylinder is detected by measuring forward scattered light and forward fluorescence. With this method, it was not always possible to accurately detect a cylinder in a sample in which formed components other than the cylinder appeared at the same time. In other words, some of the glass cylinders and the mucus threads have weak fluorescence intensity and similar lengths, which makes it difficult to discriminate them. Therefore, the forward scattered light and the forward fluorescence are measured, the electric resistance signal is further measured, and the measurement parameters of the pulse width (reflecting length information) and the resistance peak value (reflecting volume information) of scattered light are combined. This makes it possible to detect the glass column and the mucus thread with high accuracy. In addition, the cylinder containing the inclusion can be detected by combining the scattered light pulse width and the fluorescence pulse width.

[0050]

EXAMPLE An example of the reagent for analyzing formed elements in urine and the cell analysis method using the reagent of the present invention will be described.

Example 1 A diluent and a dyeing solution were prepared according to the following formulations. -Diluent Buffer HEPES 50mM NaOH pH 7.0 Amount Osmotic pressure compensating agent Sodium propionate 150mOsm / kg Amount Chelating agent EDTA-3K 0.4W / W% Electric conductivity was 5mS / cm.

Dyeing solution First dye DiOC6 (3) 400ppm Second fluorescent dye EB 1600ppm As a solvent, ethylene glycol was used.

After diluting 400 μl of urine with 1160 μl of the above dilution solution, 40 μl of the above staining solution was added (dilution ratio 4 times),
Staining was performed at ℃ for 10 seconds, and forward scattered light, forward fluorescence and electric resistance signals were measured by a flow cytometer using an argon laser as a light source. The fluorescence may be lateral (90 °) fluorescence. 1 and 2 show scattergrams obtained by measuring forward scattered light and forward fluorescence using samples in which yeast-like fungi, white blood cells and red blood cells appeared. As is clear from FIGS. 1 and 2, when the reagents of the above examples are used, yeast-like fungi, white blood cells and red blood cells are well classified. A schematic diagram of a scattergram when the formed components in urine are measured using the reagent of the present invention is shown in FIG.

Further, even when a sample having a back fluorescence is measured using the reagent of the above-mentioned example, the cells are strongly stained, so that the measurement can be performed without being influenced by the back fluorescence. . Furthermore, when the electric resistance signal is measured, a relatively large formed component such as a cylinder (a glass cylinder and a cylinder including inclusions), a mucus thread, an epithelial cell, etc. is found from the relationship between the scattered light pulse width and the resistance peak value. It was possible to catch and classify. A schematic diagram of the scattergram is shown in FIG.
Furthermore, when a column containing granules, red blood cells, white blood cells, etc. appeared among the columns, it was possible to more reliably measure the column by combining the fluorescence pulse width and the scattered light pulse width. A schematic diagram of the scattergram is shown in FIG.
Shown in

Example 2 Sodium propionate was added in an amount of 210 mOsm / kg,
The same operation as in Example 1 was repeated except that the electric conductivity was 7 mS / cm, and the same operation was repeated. The results were the same as in Example 1.

Example 3 The same operation was repeated using the reagent having the same composition as in Example 1 except that sodium propionate was added in an amount of 310 mOsm / kg and the electric conductivity was 10 mS / cm. Result is,
Same as Example 1.

Example 4 A diluent and a dyeing solution were prepared according to the following formulations.・ Diluent Buffer HEPES 50mM NaOH pH 7.0 Amount Osmotic pressure compensator NaCl 280mOsm / kg Amount Chelating agent EDTA-3K 0.4W / W%

Dyeing Solution Dye NK-529 800ppm Ethylene glycol was used as the solvent.

After diluting 400 μl of urine with 1184 μl of the above diluent, 16 μl of the above staining solution was added and stained at 35 ° C. for 50 seconds,
Forward scattered light and forward fluorescence were measured with a flow cytometer using a red semiconductor laser as a light source. The results were the same as in Example 1.

Comparative Example 1 Diluent Buffer (pH 5.0) Citric acid 27.6 mM Sodium citrate 22.4 mM Osmolarity compensating agent Sodium propionate 250 mOsm / kg amount Chelating agent EDTA-3K 0.4 W / W% Electrical conductivity The degree was 10.7 mS / cm.

Dyeing Solution Dye Auramine O 30000 ppm Solvent was ethylene glycol. After diluting 400 μl of urine with 1160 μl of the above dilution solution, add 40 μl of the above staining solution (4 times the dilution ratio), stain at 35 ° C. for 10 seconds, and use a flow cytometer with an argon laser as a light source for forward scattered light and forward fluorescence and electricity. The resistance signal was measured. 7 and 8,
The scattergram which measured the forward scattered light and the forward fluorescence is shown. As is clear from FIGS. 7 and 8, when the reagents of the above Comparative Examples were used, the yeast-like fungi and erythrocytes were poorly stained, and they could not be discriminated.

When a sample having a back fluorescence is measured using the reagent of the above comparative example, as shown in FIGS. A region that cannot be analyzed was developed and analysis was not possible.

Further, when measuring the forward scattered light and the forward fluorescence in the above-mentioned Example 1, about 1 in the case of measuring Comparative Example 1 is used.
The measurement was carried out with a detector sensitivity of 6.6%, and it was confirmed that yeast-like fungi, white blood cells and red blood cells were well classified even when a less sensitive detector was used. That is,
In Comparative Example 1, since the fluorescence of red blood cells is very weak, it is necessary to increase the detector sensitivity, but in the present example, since red blood cells are also strongly stained, a lower detector sensitivity is used. be able to. Therefore, it became possible to measure with the detector sensitivity lowered to such an extent that the background fluorescence can be ignored.

[0064]

EFFECTS OF THE INVENTION According to the reagent for analyzing formed particles in urine and the cell analysis method using the reagent of the present invention, (1) the red blood cell staining property is improved and it is possible to separate it from crystals. (2) Since the dyeability of various formed elements appearing in urine is improved, it is possible to reduce the sensitivity of Photomul (PMT) for fluorescence detection to such an extent that the influence of fluorescence behind urine itself can be ignored. became. (3) It became possible to discriminate more accurately even if various formed elements appeared in urine at the same time. (4) It was possible to improve the discrimination ability of the cylinder.

[Brief description of drawings]

FIG. 1 shows a scattergram obtained by measuring forward scattered light and forward fluorescence when dyeing urinary particles using the reagent for analyzing urinary particles of the present invention.

FIG. 2 is an enlarged view of FIG.

FIG. 3 is a schematic diagram of a scattergram when forward scattered light and forward fluorescence are measured when a urinary particle analysis reagent of the present invention is used to stain a urine particle.

FIG. 4 is a scattergram showing the relationship between the scattered light pulse width and the resistance peak value of the electrical resistance signal measured after staining the urinary particles with the reagent for analyzing the urinary particles of the present invention. It is a schematic diagram.

FIG. 5 is a scattergram showing the relationship between the scattered light pulse width and the fluorescence pulse width of the electrical resistance signal measured after staining the urinary particles with the reagent for analyzing the urinary particles of the present invention. It is a schematic diagram.

FIG. 6 is a schematic diagram showing a flow cytometer that is preferably used when measuring a urinary particle component using the reagent for analyzing a urinary particle component of the present invention.

FIG. 7 is a scattergram obtained by measuring the forward scattered light intensity and the forward fluorescence intensity when dyeing a urinary particle component using a reagent for analyzing a urinary particle component containing auramine O.

FIG. 8 is an enlarged view of FIG. 7.

FIG. 9 shows a scattergram obtained by measuring the forward scattered light intensity and the forward fluorescence intensity when a urinary formed substance analysis reagent containing auramine O was used to stain a formed component in urine having back fluorescence. .

FIG. 10 is an enlarged view of FIG.

[Explanation of symbols]

 WBC White blood cell RBC Red blood cell YEAST Yeast-like fungus

Claims (17)

[Claims] 1. A buffering agent for maintaining a pH of 5.0 to 9.0, (ii) an osmotic pressure compensating agent for maintaining an osmotic pressure of 100 mOsm / Kg to 600 mOsm / Kg, and (iii) a condensed benzene derivative system. A reagent for analyzing formed particles in urine, comprising a first dye, (iv) a second fluorescent dye capable of staining damaged cells, and (v) a chelating agent. 2. The reagent according to claim 1, wherein the electric conductivity is adjusted to 1 to 10 mS / cm. 3. The first dye is represented by the following formula: Wherein, A is -O -, - S- or -C (CH _{3) 2} -,
R is a lower alkyl group, X is halogen, Y is -CH = or -NH-, n is 0 or 1, and B is (Wherein A and R are as defined above) or a phenyl group substituted with two lower alkoxy groups or one di-lower alkylamino group (wherein this lower alkyl may be substituted with a cyano group). . ] The reagent according to claim 1 or 2, which is selected from 4. The reagent according to claim 1, wherein the second fluorescent dye is ethidium bromide or propidium iodide. 5. The chelating agent is an EDTA salt.
4. The reagent according to any one of 4. 6. A two-liquid form comprising (a) a dyeing solution containing a first dye and a second fluorescent dye, and (b) a diluting solution containing a buffering agent, an osmotic pressure compensating agent and a chelating agent. Claim 1
The reagent according to any one of 5 above. 7. The reagent according to any one of claims 1 to 6 is mixed with urine to stain a desired formed component in urine, and excitation light is applied to the stained formed component in urine. And analyzing scattered particles and fluorescence from the formed particles to analyze the formed particles in urine. 8. The method according to claim 7, further comprising measuring the electrical resistance after dyeing the formed components in urine. 9. The method according to claim 7, wherein the analytical reagent is preliminarily kept warm and then mixed with urine. 10. (I) A buffering agent for keeping the pH at 5.0 to 9.0, (II) An osmotic pressure compensating agent for keeping the osmotic pressure at 100 mOsm / Kg to 600 mOsm / Kg, (III) Excitable at a red wavelength Reagent for analysis of formed elements in urine, which comprises a complex dye and (IV) a chelating agent. 11. The reagent according to claim 10, wherein the electric conductivity is adjusted to 1 to 10 mS / cm. 12. A dye excitable at a red wavelength is represented by: [Chemical 4] Embedded image [Chemical 6] [Chemical 7] Embedded image 2. At least one selected from the group consisting of
The reagent according to 0 or 11. 13. The chelating agent is an EDTA salt.
The reagent according to any one of 0 to 12. 14. A two-liquid form comprising (a) a dyeing solution containing a dye that can be excited at a red wavelength and (b) a diluting solution containing a buffer, an osmotic pressure compensating agent and a chelating agent. 1
The reagent according to any one of 0 to 13. 15. The reagent according to any one of claims 10 to 14 is mixed with urine to stain a desired formed component in urine, and the stained formed component in urine is excited by an excitation light. Irradiate
A particle analysis method for analyzing a particle in urine by measuring scattered light and fluorescence from the particle. 16. The method according to claim 15, wherein the electrical resistance is further measured after staining the formed component in urine. 17. The method according to claim 15, wherein the analytical reagent is preliminarily kept warm and then mixed with urine.