JP3467340B2 - Urine red blood cell discriminator - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a device for analyzing particles contained in urine, and more particularly to a device for distinguishing the origin of red blood cells from urine.

[0002]

2. Description of the Related Art There are two causes of hematuria in which erythrocytes are mixed in urine. It is a medical disease such as nephritis and a urological disease such as bladder cancer, renal cancer and urolithiasis. In the case of hematuria, the cause must be sought. For example, a urological endoscopy (inserting a fiberscope through the urethra) is performed to check for cancer. This is very painful. However, if the cause of hematuria is not a cancer but a medical disease, this test is completely useless. Therefore, if the cause of hematuria can be easily known, a precise test suitable for the cause can be performed, and the efficiency of the test can be improved. This also has great financial and financial benefits.

Urine erythrocytes are derived from renal glomeruli (medical diseases)
As a method of distinguishing between non-glomerular origin (urological disease), (a) a method of distinguishing by morphology of erythrocytes using a microscope, (b) difference of erythrocyte size using an automatic hemocytometer Is known (the size of red blood cells derived from renal glomeruli is small).

[0004]

The method (a) has a problem that the work for inspection is complicated and takes time, and
Since it is based on human visual judgment, it is difficult to maintain reliability and requires skill.

In the method (b), it is necessary to measure the electric conductivity of the urine sample while keeping it constant, and there is a problem that pretreatment such as centrifugation is required in advance, and other than red blood cells such as bacteria and crystal components. There is a problem that an object is erroneously detected as an erythrocyte and the determination accuracy is lowered.

The present invention has been made in consideration of such circumstances, and the origin of urinary erythrocytes can be accurately determined by only processing scattered light of particles obtained from a flow cytometer and optical signals of fluorescence intensity signals. A device that can be easily identified is provided.

According to the present invention, a sheath flow cell for forming a urine sample flow by wrapping a fluorescent-stained urine sample in a sheath liquid, a light irradiation means for irradiating the urine sample flow with light, and each particle in the urine sample flow. Light detection means for detecting scattered light and fluorescence emitted by the erythrocyte, identification means for identifying red blood cells from particles in the urine sample flow based on the detected fluorescence , and particle size distribution of red blood cells based on the scattered light of the identified red blood cells A urine erythrocyte discrimination apparatus comprising: a particle size distribution creating means for creating a diagram; and a determining means for determining the origin of red blood cells based on the biased state of the particle size distribution chart created based on scattered light .

The urine sample in the present invention is, for example, a sample obtained by diluting raw urine or urine with a diluent and then adding a fluorescent dye.

The sheath flow cell for forming a urine sample flow is a flow cell capable of forming a thin sample flow by a hydrodynamic effect by wrapping a urine sample in a sheath liquid and flowing it. Can be used.

The light irradiation means for irradiating the urine sample stream with light is
It is a means for continuously irradiating the urine sample stream with light, which can be, for example, a light source such as a laser, a halogen lamp or a tungsten lamp.

The particles in the urine sample stream are formed elements contained in urine, that is, red blood cells, white blood cells, epithelial cells, bacteria, yeast-like fungi and sperm. Examples of the light detecting means for detecting the intensity of the light emitted by the particles include a photodiode or a phototransistor as the light detecting means for detecting the intensity of scattered light, and a photomultiplier as the light detecting means for detecting the intensity of fluorescence. Can be used.

The identification means for identifying red blood cells from the particles in the urine sample flow is, for example, a two-dimensional scattergram (distribution chart) created using the scattered light intensity and fluorescence intensity detected from each particle as parameters, The particles distributed within a predetermined distribution area are determined as red blood cells.

That is, it is known that the particles in the urine sample have the characteristics of the scattered light intensity and the fluorescence intensity as shown in Table 1. Therefore, on the scattergram,
Those contained in the regions of scattered light intensity 40 to 140 and fluorescence 20 to 30 can be identified as red blood cells. The method for identifying red blood cells is not limited to this.

[Table 1]

The particle size distribution creating means for creating a particle size distribution map of red blood cells based on the identified light signal intensity of the red blood cells is a property in which scattered light intensity, especially forward scattered light intensity, corresponds to the cross-sectional area (particle size) of the particle. Therefore, a histogram with the scattered light intensity as a parameter is created as a particle size distribution chart. The side scattered light intensity is susceptible to the shape or surface state of red blood cells, but it can be used instead of the forward scattered light.

Further, in the determining means for determining the origin of red blood cells based on the biased state of the particle size distribution map, determining the origin of red blood cells means identifying the source of red blood cells in urine, that is, whether they are derived from glomeruli. Is to judge. This judgment uses the characteristic that red blood cells derived from glomeruli are generally smaller in particle size than non-glomerular ones. Non-glomerular origin type is biased to, and intermediate type is classified as mixed type of both. Specifically, an index that reflects the biased state of the particle size distribution of red blood cells is calculated, and the value is used for determination.

Preferably, the judging means has a lower limit value La, an upper limit value Lz, and a set value L in the particle size distribution.
1 and L2 (La <L1 <L2 <Lz), La
And Rz, the number of red blood cells Raz, the number of red blood cells Ra2 between La and L2, and the number of red blood cells R1z between L1 and Lz.
The origin of red blood cells is determined from the values of 2 / Raz and R1Z / Raz.

That is, the determination means determines that red blood cells are of glomerular origin if Ra2 / Raz is greater than or equal to the first predetermined value, and is non-glomerular origin if R1z / Raz is greater than or equal to the second predetermined value. judge.

More specifically, the particle size is, for example, La = 0, L1 = 4 μm, L2 = 6 μm, Lz = 10 μ in terms of particle size.
m is set, and if Ra2 / Raz ≧ 0.8, it is determined that the red blood cells are of glomerular origin, and if R1z / Raz ≧ 0.8, it is determined that the red blood cells are of non-glomerular origin. If not, it is judged as mixed type. The set numerical value is not limited to this and can be set arbitrarily. Further, the determination means may be a simple means provided with a calculation means for calculating an average value of particle sizes in the particle size distribution and determining the origin of red blood cells from the calculated value.

The identifying means, the particle size distribution creating means, and the determining means can be constituted by a microcomputer including a CPU, ROM and RAM.

[0020]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail with reference to the embodiments shown in the drawings. This does not limit the invention. Configuration of Red Blood Cell Differentiation Device FIG. 1 is a configuration diagram showing the configuration of an optical system of a flow cytometer which is the urine red blood cell discrimination device of the present invention, and FIG. 2 is a block diagram showing the configuration of a signal processing system thereof. In FIG. 1, the sheath flow cell 1 forms a urine sample flow 2 by wrapping a urine sample that has been subjected to a fluorescent staining process in advance in a sheath liquid.

The light emitted from the argon laser light source 3 illuminates the particles 4 of the urine sample flow 2. The light that directly passes through the urine sample flow 2 is blocked by the beam stopper 5, and the forward scattered light and the forward fluorescence emitted by the particles 4 are collected by the condenser lens 6,
The forward scattered light is reflected by the dichroic mirror 7 and detected by the photodiode 8. On the other hand, the forward fluorescence passes through the dichroic mirror 7 and is detected by the photomultiplier 9.

In FIG. 2, the detection signals S1 and S2 of the photodiode 8 and the photomultiplier 9 are input to the signal processing device 10, respectively. Signal processing device 10
Processes the signals S1 and S2 in accordance with various setting conditions input from the input device 11, and displays the processing result on the display device 1
Display on 2.

Here, the signal processing device 10 includes a CPU, an R
The input device 11 is composed of, for example, a keyboard and a mouse, and the display device 12 is composed of a CRT and a liquid crystal display.

The basic operation of such a configuration will be described with reference to the flowchart of FIG. Step 101 in FIG.
In the first step, a urine sample prepared by first diluting urine with a diluting solution and then adding a dyeing solution is supplied to the sheath flow cell 1 together with the sheath liquid to form a urine sample flow 2.

At the same time, the urine sample flow 2 is irradiated with laser light from the light source 3, and the forward scattered light and the forward fluorescence emitted by each particle are respectively fed to the photodiode 8 and the photomultiplier 9.
The forward scattered light intensity and the forward fluorescence intensity detected in step S10 are stored in the RAM in the signal processing device 10 (step 10).
2).

Next, the signal processing device 10 creates a two-dimensional scattergram having the forward fluorescence intensity and the forward scattered light intensity as parameters and displays it on the display device 12. Next, when the user gives an instruction for red blood cell identification processing using the input device 11, particles belonging to a predetermined area of the two-dimensional scattergram are identified as red blood cells (step 104).

Next, a particle size distribution map of the identified red blood cells, here a histogram with the forward scattered light intensity as a parameter, is created and displayed on the display device 12 (step 10).
5). The user uses the input device 11 to set the lower limit value La, the upper limit value Lz, and the set values L1 and L2 for the forward scattered light intensity.
When (La <L1 <L2 <Lz) is set (step 1
06), the number of red blood cells Raz between La and Lz in the histogram, the number of red blood cells Ra2 between La and L2, and the number of red blood cells R1z between L1 and Lz
And Ra2 / Raz and R1z / Raz are respectively calculated (step 107).

Next, set values Q1 and Q2 are input from the input device 11.
When Ra2 / Raz ≧ Q1, red blood cells are determined to be glomerular-derived type, and when R1z / Raz ≧ Q2, red blood cells are determined to be non-glomerular-derived type. The determination result is displayed on the display device 12 (step 109).

Implementation of differentiation of erythrocytes 98 cases (16-75) in which bleeding sites in urine are known and bacteriuria is not observed by microscopic observation
Age), 64 males, 34 females, and 100 or more erythrocytes in the histogram of this device.

[0030] A case with glomerular disease was confirmed to have no abnormality in the urinary tract after transvenous nephroureterography or ultrasonic examination, and had some glomerular disorder by renal biopsy.
Cases with non-glomerular diseases were cases of transvenous nephroureterography, cystoscope, histological diagnosis confirmed urological abnormalities, cases after transurethral surgery and prostate,
We selected 67 cases with bleeding from the bladder and 67 cases with apparent urological hematuria after lithotripsy due to extracorporeal shock waves due to urinary tract stones (Table 1).

For these cases, the intermediate urine was put into a glass or plastic spitz immediately after the urine collection, and the measurement was performed within 6 hours after the collection. At the time of measurement, 400 μl of the collected urine was diluted with 1160 μl of the diluting solution, 40 μl of the staining solution was added (dilution ratio of 4 times), and the cells were stained at 35 ° C. for 10 seconds, and the forward flow cytometer shown in FIGS. Scattered light and forward fluorescence were measured.

The diluting solution and the dyeing solution were prepared according to the following formulations.・ Diluent Buffer HEPES 50mM NaOH pH 7.0 Amount Osmotic pressure compensator Sodium propionate 150mOsm / kg Amount Chelating agent EDTA-3K 0.4w / w% ・ Dyeing liquid 1st dye DiOC6 (3) 400ppm 2nd Fluorescent dye EB 1600ppm

Then, erythrocytes are identified from particles in urine, and it is examined whether or not it is possible to distinguish between glomerular type and non-glomerular type based on the difference in the frequency distribution (particle size distribution) of the forward scattered light in this flow cytometer. did. In 69 cases, 100 ml of urine was spun down at 1500 rpm for 5 minutes using an ordinary light microscope (Model S manufactured by Nippon Kogaku Co., Ltd.), and 100 specimens of the same examiner were examined 400 times at 400 times without staining. The origin was determined. All verdicts were blind and blind to the clinical diagnosis.

Based on the above, the sensitivity and specificity of the clinical diagnosis, the judgment by this apparatus, and the visual judgment result by an optical microscope were detected, and their usefulness was examined. Sensitivity is the concordance rate of clinical diagnosis and positive results, and it was judged that those showing a mixed type were included in positives as described later. Specificity means the negative rate of the result in people who do not have the disease.

FIG. 4 shows an example of a two-dimensional distribution chart (scattergram) of the "fluorescence-scattered light" intensity displayed on the display device 12 of this apparatus. FSC represents the cross-sectional area of cells by the forward scattered light intensity, and FL represents the fluorescence staining intensity. DN for nuclear staining
A, reflects the amount of RNA. Membrane staining is also carried out at the same time because it is not possible to distinguish red blood cells from those without crystals and nuclei by nuclear staining alone. In this way, it is possible to clearly separate red blood cells, white blood cells, epithelial cells, bacteria, fungi, casts, and crystal components from the particle size and fluorescence intensity, and the respective numbers can be measured.

FIG. 5 shows a typical histogram obtained by analysis of the present apparatus derived from glomerulus and non-glomeromer. The non-glomerular type hematuria had the center and peak of the distribution in the larger size, and the glomerular type had the center and peak of the distribution smaller. In the former, the peak of the distribution was steeper, and most of them showed a large size. The latter showed a more chronic distribution from small to large. That is, there was an overlapping area (overlap zone) between the two.

Therefore, as shown in FIGS. 6 and 7, in non-glomerular type clinical diagnosis in individual cases, the cumulative frequency distribution occupies 80% of the larger size (non-glomerular type discrimination point) L1, thread On the other hand, in the case of the spherical type, the point (glomerular type identification point) L2 occupying 80% of the smaller size was determined.
The reason why 80% is adopted here is that the diagnosis using the optical microscope and the laser microscope in the past adopted the number of 80% as a reference and obtained a good determination result. These cases were plotted as shown in FIG. 8 and their patterns were compared. Among the cases of non-glomerular hematuria, 90% or more of the points that can be diagnosed were non-glomerular type identification points of 84 or less, and in the glomerular type, the glomerular type identification points of 126 or more.

The FSC intensity 84-126 was used as the above-mentioned overlap zone. Converted to red blood cell size, FSC
The intensity 84 was 4 μm and the intensity 126 was 6 μm. In the subsequent examination, 80% or more erythrocytes were present from the larger size to the FSC intensity 84 (lower limit of the overlap zone) (non-glomerular type identification point was 84 or more) was non-glomerular type and small in size. On the other hand, those having 80% or more by 126 (upper limit of the overlap zone) (glomerular type identification point is 126 or less) were judged to be glomerular type, and those showing 80% or less were judged to be mixed type. In addition, cases including 80% or more in all cases including the laser microscope so far were determined to be non-glomerular type.

Table 2 shows the results determined by this apparatus and the clinical diagnosis results for each case. 28 out of 31 glomerular cases were derived from glomeruli, 3 were judged to be mixed hematuria, 62 out of 67 urological diseases were judged to be non-glomerular and 5 were judged to be mixed. According to the determination by this device, the sensitivity seen from glomerular disease was 100% and the specificity was 92.54% (Table 3).

[Table 2]

The sensitivity to glomerular disease was 95.83% and the specificity was 93.33% as determined by visual inspection using an optical microscope (Table 4). In addition, the sensitivity of this device to glomerular disease based on the visual determination result is 100%, and the specificity is 9
It was 3.02% (Table 5).

[Table 3]

[Table 4]

[Table 5]

In creating the diagnostic criteria, the criteria are created by deliberately excluding the cases showing bacteriuria by observation with an optical microscope. In bacteriuria, the experience of urinary erythrocyte observation with a laser microscope sometimes showed glomerular shape and appearance of miniaturized erythrocytes.

In the discrimination method of urinary erythrocytes derived from a laser microscope, there was a difference that the non-glomerular and glomerular-derived erythrocytes were located at positions of almost the same size (overlap zone). From experience, it was considered to be around 5 μm. When urinary erythrocytes were judged by a laser microscope, they were judged by looking at the size and shape, but in this example, the device determines the origin only by the cross-sectional area, that is, the size of the erythrocytes. It was considered necessary to clarify and determine the overlap zone to determine the diagnostic criteria.

Examination of spherical latex particles of various sizes measured by this apparatus and the FSC strength, 1 μm corresponds to about 21. This time, the size of red blood cells measured (FSC intensity)
The diagnostic standard was created based on where 80% of the red blood cell numbers are distributed (discrimination point). FSC strength 84
-126 is 4-6 μm when converted to red blood cell size
Met. The overlapping zone was considered to have a size that can belong to both glomerular and non-glomerular erythrocytes.

The non-glomerular type is not only the identification point of 84 or more in the part of the preparation of the diagnostic standard, but also the non-glomerular type which shows 80% or more in both measurement from the larger size and the smaller size. The reason for using a spherical shape will be described. Both 80%
Showing the above means that red blood cell size is concentrated in the overlap zone.

That is, in the case of a non-glomerular sample containing a large amount of red blood cells in urine, many red blood cells showing a Competito shape or shrinking to about 5 μm without being deformed are observed. There is a tendency for the size to be about 5 μm, that is, to gather in the overlap zone. This is a fact that I noticed when I was preparing a method for identifying hematuria with a laser microscope.

This is similar to an image in which erythrocytes are transformed into Competo within a few minutes when venous blood is dropped on a slide glass and observed with a microscope. Unlike red blood cells derived from glomeruli that are deformed in the urine, the erythrocytes that have been transformed into Compeit's urine are almost uniform in the projections when observed with one red blood cell, and can be easily distinguished by a laser microscope. Tsuku.

There was one sample that showed 80% or more in both cases when examined with this device, but the observation with a microscope showed the above-mentioned characteristics, and the size distribution was such that most red blood cells were distributed in the overlap zone. It was a spherical case. In addition, even in the glomerular cases in which the examiner of the visual judgment recognized that there were many relatively large deformed red blood cells, none of them showed a non-glomerular type in the ratio of the particle size distribution in this device.

The measurement with this apparatus showed a sensitivity of 100% and a specificity of 92.54%. This was almost the same as the discrimination method using a laser microscope. The examiner who made the visual judgment this time was very skilled in the discrimination of urine erythrocytes, and the sensitivity and specificity of the visual judgment were also high. It is considered that the correlation between the result and the determination result by this device is high and that the urinary erythrocyte-derived discrimination method by this device is very reliable.

Next, a comparison between this device and an automatic blood cell counter will be described. The number of erythrocytes in urine for which the particle size distribution can be visualized with an automatic hemocytometer is about 500-9000 / μl. If the number is higher than this, urine dilution is necessary. It is known that a sediment operation needs to be added. This method is not suitable for multi-sample treatment in a clinical setting because it requires time-consuming centrifugation of urine and resuspension in a dedicated liquid, which is troublesome. In addition, there is a drawback in that urinary components having a size equal to or smaller than that of red blood cells are also regarded as red blood cells.

Next, the clinical significance of this device will be described. Conventionally, hematuria derived from urological disease or hematuria of renal glomerular disease, depending on the urinary erythrocyte morphology, before performing cystoscopy and transvenous nephroureterography, which are invasive for patients with microscopic hematuria. It is suggested that these should be examined microscopically before their urological examination.

The present inventor examined the disease of microscopic hematuria at the urological examination using a real-time laser microscope, and found that 80% was glomerular hematuria. As described above, the majority of patients with microscopic hematuria have glomerular disease, and depending on the test results of this device, transvenous nephroureterography,
It is possible to omit examinations necessary for urological diseases such as ultrasonic examination, cystoscope, and urine cytology, and it is considered preferable from the viewpoints of patient invasion and medical economy.

In the case of children, there are many cases of asymptomatic cases, and it is often the case that renal disease is not detected for the first time in school urinalysis, and the usefulness of school urinalysis is discussed. With the conventional method, various tests are required as with adults. However, if this device is used at the time of secondary urinalysis, it is possible to noninvasively determine whether hematuria is derived from glomeruli or non-glomeruli, and unnecessary tests can be omitted in subsequent tests, which is a physical examination of children. The burden will be greatly reduced.

To summarize the above, 1) In this device, red blood cells derived from glomeruli were distributed in a smaller size, and larger blood cells derived from a non-glomerular body were distributed, and it was possible to distinguish them by size. However, there is an overlapping zone where the size overlaps between them, and the FSC intensity is 84 to 126.
m to 6 μm.

2) Diagnostic criteria were created based on the above facts. 80% or more of non-glomerular type with 80% or more erythrocytes from the larger size to FSC intensity 84 (lower limit of overlap zone), and 126% from the smaller size to 126 (upper limit of overlap zone) Those that were present were judged to be glomerular type, and those showing 80% or less were judged to be mixed type. The cases showing 80% or more were judged to be non-glomerular type. Based on the above diagnostic criteria, 98 (31 are glomerular diseases, 67 are urological diseases) hematuria was differentiated, and sensitivity to glomerular disease was 100% and specificity was 9
It showed 2.54%.

[0055]

According to the device of the present invention , red blood cells in urine
It is possible to easily determine the origin of erythrocyte, and it is possible to put it into practical use for the differential diagnosis of hematuria due to the fact that a large amount of samples can be processed in a short time, and special technology and knowledge are not required for the determination.

[Brief description of drawings]

FIG. 1 is a structural explanatory view showing an optical system of an apparatus of an example.

FIG. 2 is a block diagram showing a signal processing system of the apparatus of the embodiment.

FIG. 3 is a flowchart showing a basic operation of the embodiment.

FIG. 4 is an explanatory diagram showing a display example of the embodiment.

FIG. 5 is a histogram showing an example of measurement results obtained by the apparatus of the example.

FIG. 6 is an explanatory diagram showing a method of distinguishing red blood cells using a histogram of the example.

FIG. 7 is an explanatory diagram showing a method for identifying red blood cells using a histogram according to an example.

FIG. 8 is a graph in which glomerular type identification points and non-glomerular type identification points are plotted for all samples of the examples.

[Explanation of symbols] 1 Sheath flow cell 2 Urine sample flow 3 light sources 4 particles 5 beam stopper 6 Condensing lens 7 dichroic mirror 8 photodiodes 9 Photomultiplier

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor, Satoshi Sakai 1-15-1, Kitasato, Sagamihara-shi, Kanagawa Kitasato University Hospital Kidney Center (56) Reference JP-A-5-322883 (JP, A) JP-A 4-337460 (JP, A) Toshiharu Okada, et al., Clinical Pathology, 1989, Volume 37, No. 1, pp. 67-72 Hiromura Imura, et al., Sairin Gikai, 1991, Vol. 38, No. 1, Pages 22-26 (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 15/14 G01N 15/02 G01N 33/493

Claims (5)

(57) [Claims] 1. A sheath flow cell for wrapping a fluorescent-stained urine sample in a sheath liquid to form a urine sample flow, a light irradiation means for irradiating the urine sample flow with light, and each particle in the urine sample flow is released. A light detection means for detecting scattered light and fluorescence, an identification means for identifying red blood cells from particles in the urine sample flow based on the detected fluorescence , and a particle size distribution map of red blood cells based on the scattered light of the identified red blood cells. Particle size distribution creation means to create and scattering
A urinary erythrocyte discrimination apparatus comprising: a determination unit that determines the origin of erythrocytes based on the biased state of the particle size distribution chart created based on light . 2. The determining means in the particle size distribution has a particle size lower limit value of La, an upper limit value of Lz, and set values of L1 and L2.
When (La <L1 <L2 <Lz), the number Raz of red blood cells belonging to La and Lz, the number Ra2 of red blood cells belonging to La and L2, and the number R1z of red blood cells belonging to L1 and Lz. Ra2 / Ra is provided with a calculating means for calculating
The urine erythrocyte discrimination apparatus according to claim 1, wherein the origin of erythrocytes is determined from the values of z and R1z / Raz. 3. The determination means determines that red blood cells are of glomerular origin if Ra2 / Raz is greater than or equal to a first predetermined value and R
The urinary erythrocyte differentiation apparatus according to claim 2, wherein if 1z / Raz is equal to or more than a second predetermined value, it is determined to be a non-glomerular origin type. Wherein the constant means, characterized in that the identification to create a two-dimensional distribution diagram using the scattered light intensity and a fluorescent intensity detected from each particle, the particles distributed in a predetermined distribution area as erythrocytes The urine erythrocyte discrimination apparatus according to claim 1. 5. The urinary erythrocyte discrimination apparatus according to claim 1, wherein the determination means determines the origin of the red blood cell as one of a glomerular origin type, a non-glomeromer origin type and a mixed type.