JP4413051B2 - Fluorescent particle discrimination device, fluorescent particle counting device equipped with the fluorescent particle discrimination device, and fluorescent particle discrimination method - Google Patents

Description

    The present invention relates to a fluorescent particle discriminating device that discriminates whether or not a fluorescent particle to be measured is a specific fluorescent particle, a fluorescent particle counting device including the fluorescent particle discriminating device, and a fluorescent particle discriminating method.

    Conventionally, fluorescence-stained particles are irradiated with excitation light to determine the type of particles and to measure the number of particles.

    For example, in the medical field, platelet preparations and red blood cell preparations obtained by extracting platelets and red blood cells from blood are used, but it is preferable that no white blood cells remain in these platelet preparations or red blood cell preparations. Leukocyte removal is performed. Various white blood cell counting methods for confirming whether white blood cells have been removed from platelet preparations or red blood cell preparations have been proposed (see, for example, Patent Document 1).

    Here, FIG. 12 is a block diagram showing an example of the structure of a conventional apparatus for counting white blood cells, and FIG. 13 is a flowchart showing an example of a white blood cell counting method. Reference numeral 100 in FIG. 12 indicates an imaging container for containing a platelet preparation or the like, reference numeral 101 indicates a laser light source for irradiating the imaging container 100 with a laser beam, reference numeral 102 indicates a mirror, reference numeral 103 Indicates a CCD camera.

    In order to count the number of leukocytes in blood, platelet preparations or erythrocyte preparations using such a device, first put a drug solution (surfactant such as Triton X) into the blood or platelet preparations, etc. Other than the above (such as platelets and red blood cells) are removed (see S21 in FIG. 13), and then white blood cells are fluorescently stained with a fluorescent dye such as propidium iodide (see S22). When the pre-treated platelet preparation or the like is placed in the above-described imaging container 100 and irradiated with a laser beam (excitation light) by the laser light source 101 (see S23), a white blood cell image is taken by the CCD camera 103. (Refer to S24). The image is captured by a computer, subjected to image processing, and the number of white blood cells is counted (see S25).

JP-A-11-183382

    By the way, when contaminants such as dust and dust are mixed in blood etc., they remain without being dissolved by the surfactant, so it is difficult to distinguish white blood cells from contaminants, There was a problem that the white blood cell counting accuracy deteriorated.

Further, the fluorescent image signal of leukocytes has a distribution shape generally indicated by f ₁ (x) in FIG. 14, and the fluorescent image signal of impurities is, for example, a distribution shape indicated by f ₂ (x) in FIG. and it has, but distinguished as attempt to distinguish the two in comparison with the threshold value indicated by reference numeral C ₁ is difficult and, if you try to distinguish between the two compared to the threshold value indicated by reference numeral C _2, thresholds too low There was a problem of being easily affected by noise.

    An object of the present invention is to provide a fluorescent particle discriminating apparatus capable of discriminating fluorescent particles with high accuracy, a fluorescent particle counting apparatus including the fluorescent particle discriminating apparatus, and a fluorescent particle discriminating method.

The invention according to claim 1 is illustrated in FIG. 1, and includes an excitation light source (3) for irradiating accommodated fluorescent particles with excitation light, and fluorescence generated by irradiation of the excitation light. In the fluorescent particle discriminating device (1) configured to provide a fluorescence measuring means (4) for capturing and measuring the intensity distribution,
Differentiating means for outputting differential signals (f ′ (x, y), f ′ (x)) obtained by differentiating the intensity distribution signals (f (x, y), f (x)) from the fluorescence measuring means (4). (5) and
Based on adding the differential signal (f ′ (x, y), f ′ (x)) to the intensity distribution signal (f (x, y), f (x)), the peak portion and the peak portion are combined signal having a valley on the outer side of the rising portion a generally mountain-shaped consisting of a rising portion (reference symbol G ₁ in FIG. 3 (c)) continuing (g (x, y), g (x)) Adding means (6) for outputting
The combined signal (g (x, y), g (x)) values first threshold value (Fig. 3 reference numeral references C ₁ of (c)) the first threshold based on the fact to determine whether it exceeds a (C ₁ ) peak portion detection means (7) for detecting the peak portion exceeding,
The value of the composite signal (g (x, y), g (x)) from the side close to the peak part detected by the peak part detection means (7) to the side close to the valley part (G ₁ ) Distribution region of the rising portion exceeding the second threshold value (C ₂ ) based on sequentially determining for each minute portion whether or not exceeds the second threshold value (see symbol C _{2 in} FIG. 3C) Rising portion detection means (8) for detecting the magnitude of
A reference value generating means (15) for generating a reference value serving as a reference when determining whether the particle is a specific fluorescent particle;
Fluorescent particle discrimination for discriminating whether or not the fluorescent particle being measured is a specific fluorescent particle based on the detection result of the rising portion detection means (8) and the reference value from the reference value generation means (15) And means (9).

The invention according to claim 2 is the invention according to claim 1, wherein the peak portion detection means (7) detects the size of the distribution region of the peak portion,
The fluorescent particle discriminating means (9) includes a size of the distribution area of the rising part detected by the rising part detection means (8), and a size of the distribution area of the peak part detected by the peak part detection means (7). Then, based on the reference value from the reference value generating means (15), it is determined whether or not the fluorescent particles being measured are specific fluorescent particles.

In the invention according to claim 3, as illustrated in FIG. 3 (c), in the invention according to claim 1 or 2, the second threshold value (C ₂ ) is smaller than the first threshold value (C ₁ ). And the value of the combined signal (g (x, y), g (x)) in the outer portion of the valley (G ₁ ) is substantially equal.

As illustrated in FIG. 1, in the invention according to claim 4, in the invention according to any one of claims 1 to 3, the peak detection unit (7) sets the first threshold value (C ₁ ). First threshold value generating means (70) for outputting and a first threshold value for determining whether or not the value of the composite signal (g (x, y), g (x)) exceeds the first threshold value (C ₁ ). Comparing means (71),
The first threshold generation means (70) calculates and outputs the first threshold (C ₁ ) according to the illumination state of the excitation light with respect to the fluorescent particles.

As illustrated in FIG. 5, the invention according to claim 5 is the invention according to any one of claims 1 to 4, wherein the composite signal (g (x, y) is obtained for a predetermined region including the peak portion. ), G (x)) memory means (13) for storing values,
Whether the rising portion detecting means (8) has a value of the combined signal (g (x, y), g (x)) stored in the memory means (13) exceeding a second threshold value (C ₂ ). The size of the distribution region of the rising portion exceeding the second threshold value (C ₂ ) is detected based on sequentially determining whether each minute portion from the side close to the peak portion. .

The invention according to claim 6, as illustrated in FIG. 1, is the fluorescent particle discrimination device (1) according to any one of claims 1 to 5;
The present invention relates to a fluorescent particle counting device characterized by comprising a fluorescent particle counting means (10) for counting the number of specific fluorescent particles based on the determination result by the fluorescent particle determining device (1).

The invention according to claim 7 irradiates the fluorescent particles with excitation light;
Capturing fluorescence generated based on irradiation of the excitation light and measuring its intensity distribution;
Differentiating the intensity distribution signal (f (x, y), f (x)) obtained by the measurement and outputting a differential signal (f ′ (x, y), f ′ (x));
Based on adding the differential signal (f ′ (x, y), f ′ (x)) to the intensity distribution signal (f (x, y), f (x)), the peak portion and the peak portion are A step of outputting a composite signal (g (x, y), g (x)) having a substantially mountain shape composed of continuous rising portions and having a valley portion (G ₁ ) outside the rising portion;
The combined signal (g (x, y), g (x)) the peak value of is greater than the first threshold value (C ₁₎ based on the fact to determine whether it exceeds a first threshold value (C ₁₎ Detecting a part;
From the side close to the peak part to the side close to the valley part (G ₁ ), the value of the combined signal (g (x, y), g (x)) exceeds the second threshold (C ₂ ). Detecting the size of the distribution region of the rising portion that exceeds the second threshold (C ₂ ) based on sequentially determining whether or not each minute portion;
And a step of determining whether or not the fluorescent particle being measured is a specific fluorescent particle based on a detection result and a reference value regarding the size of the distribution region of the rising portion. .

    The numbers in parentheses are for the sake of convenience indicating the corresponding elements in the drawings, and therefore the present description is not limited to the descriptions on the drawings.

    According to the first and seventh aspects of the invention, the determination as to whether or not the second threshold value is exceeded is performed only for the portion from the side close to the peak portion to the side close to the valley portion. Since it is not performed for the portion outside the portion, the influence of noise outside the valley portion can be avoided, and the fluorescent particles can be accurately identified.

    According to the second aspect of the present invention, the determination by the fluorescent particle determining means is performed not only based on the size of the distribution region of the rising portion but also based on the size of the distribution region of the peak portion. Therefore, the discrimination accuracy can be improved.

    According to the invention of claim 3, since the second threshold value is smaller than the first threshold value and substantially equal to the value of the combined signal in the outer portion of the valley, Accordingly, it is possible to accurately detect white blood cells and contaminants.

    According to the invention of claim 4, since the first threshold value generating means calculates and outputs the first threshold value corresponding to the illumination state of the excitation light with respect to the fluorescent particles, the fluorescence is emitted even if the illumination state is not constant. Particles can be accurately identified.

    According to the invention of claim 5, the rising portion detecting means is configured to detect the size of the distribution area of the rising portion based on the value of the synthesized signal stored in the memory means. The rising portion detection process based on the second threshold value can be performed in a form in which the combined signal stored in the memory means is stored, and the rising edge detection process can be repeatedly performed with the second threshold value changed. Thereby, even when the second threshold value set first is not appropriate, the processing can be continued without performing the calculation of the composite signal again.

    According to the invention of claim 6, the fluorescent particle counting means can accurately count the fluorescent particles.

    Hereinafter, the best mode for carrying out the present invention will be described with reference to FIGS. 1 to 11.

    FIG. 1 is a block diagram showing an example of the structure of a fluorescent particle discriminating device and a fluorescent particle counting device according to the present invention, and FIG. 2 (a) is a diagram showing an example of the fluorescence intensity distribution (two-dimensional distribution) of white blood cells. FIG. 2B is a diagram showing an example of the fluorescence intensity distribution (two-dimensional distribution) of impurities. Fig. 3 (a) is a diagram showing an example of the fluorescence intensity one-dimensional distribution (intensity distribution signal) of the fluorescent particles, and Fig. 3 (b) is a derivative obtained by differentiating the intensity distribution signal of Fig. 3 (a). FIG. 3 (c) is a diagram showing a combined signal obtained by adding the differential signal shown in FIG. 3 (b) to the intensity distribution signal shown in FIG. 3 (a). FIG. 4 is a waveform diagram for explaining the difference between the synthetic signal of leukocytes and the synthetic signal of contaminants, and FIG. 5 shows the structure of the fluorescent particle discriminating apparatus and fluorescent particle counting apparatus according to the present invention. FIG. 6 is a block diagram for explaining a fluorescent particle discrimination method, and FIG. 7 is a schematic diagram for explaining a fluorescent particle discrimination method. FIG. 8 is a flowchart for explaining an example of the counting method of fluorescent particles, FIG. 9 is a block diagram showing an example of the structure of the fluorescent particle discriminating device according to the present invention, and FIG. It is a block diagram for demonstrating the detailed structure. Furthermore, FIG. 11 is a figure for demonstrating the effect of this invention.

The fluorescent particle discriminating apparatus according to the present invention includes an excitation light source 3 for irradiating the fluorescent particles contained therein with excitation light, as indicated by reference numeral 1 in FIG. Fluorescence is generated by irradiation with such excitation light, but the fluorescence is measured by the fluorescence measuring means 4 and its intensity distribution, that is,
A two-dimensional distribution as shown by f ₁ (x, y) and f ₂ (x, y) in FIGS. 2 (a) and (b), or
-It is configured to measure a one-dimensional distribution as shown by f (x) in FIG. In addition, the code | symbol 2 of FIG. 1 shows the fluorescent particle accommodating means for accommodating fluorescent particles (for example, leukocytes).

Moreover, the fluorescent particle discriminating apparatus 1 according to the present invention, as shown in FIG.
A differentiating means 5 for outputting a differential signal f ′ (x, y) or f ′ (x) obtained by differentiating the intensity distribution signal f (x, y) or f (x) from the fluorescence measuring means 4;
A synthesized signal g (x, y) or g (based on adding the differential signal f ′ (x, y) or f ′ (x) to the intensity distribution signal f (x, y) or f (x) adding means 6 for outputting x),
It has. Here, the synthesized signals g (x, y) and g (x) are signals as exemplified in FIG. 3C, and are composed of a peak part and a skirt part (rising part) connected to the peak part. has a Ryakuyama type, has a valley G ₁ is outside the該裾portion (i.e., outside of the composite signal).

Furthermore, the fluorescent particle discriminating apparatus 1 according to the present invention, as shown in FIG.
The first threshold value C ₁ is determined based on determining whether the value of the composite signal g (x, y) or g (x) exceeds the first threshold value (see reference symbol C _{1 in} FIG. 3 (c)). a peak portion detecting means 7 for detecting the peak portion has exceeded (reference numeral D ₁ of the FIG. 3 (c)),
- from the side adjacent to the peak portion detected by the peak portion detecting means 7 to the side close to the valley G _1, the combined signal g (x, y) or the value of the second threshold value of g (x) ( hem of detecting the 3 or exceeds the code reference C ₂₎ if the size of the distribution region of the skirt exceeding the second threshold value C ₂ based on the sequentially determined for each minute portion of (c) Part detecting means (rising part detecting means) 8;
A reference value generating means 15 for generating a reference value serving as a reference when discriminating whether the particle is a specific fluorescent particle (for example, white blood cell);
Fluorescent particle discrimination for discriminating whether or not the fluorescent particle being measured is a specific fluorescent particle based on comparing the detection result of the skirt detecting means 8 with the reference value from the reference value generating means 15 Means 9;
It has.

    In the present specification, the “minute portion” means a region having a minute area (in the figure, one rectangular portion) in the case of a two-dimensional distribution as shown in FIG. 2, as shown in FIG. In the case of such a one-dimensional distribution, it means a section having a minute length (section divided by a scale on the x-axis). Further, the peak portion detection means 7 is configured to detect at least the position of the peak portion (distributed position).

    Here, the operation of the fluorescent particle discriminating apparatus 1 will be described with reference to FIG.

Now, it is assumed that the fluorescent light indicated by the symbol A in FIG. 3 is irradiated with excitation light, and the fluorescence measuring means 4 measures the fluorescence intensity distribution indicated by the one-dimensional intensity distribution signal f (x) (FIG. See)). The differentiating means 5 differentiates the intensity distribution signal f (x) to calculate a differential signal f ′ (x) (see FIG. 5B), and the adding means 6 receives the one-dimensional intensity distribution signal f ( A synthesized signal g (x) obtained by synthesizing x) and the differential signal f ′ (x) is calculated (see FIG. 10C). Then, the peak portion detecting means 7, the combined signal g values of (x) is based on the fact to determine whether it exceeds a first threshold value C _1, the peak portion D which exceeds the first threshold value C _{1 1} and the skirt detecting means 8 detects that the value of the composite signal g (x) has a second threshold value C ₂ from the side close to the peak portion D ₁ to the side close to the valley portion G _1. The size of the distribution area of the skirt portion exceeding the second threshold C ₂ (the size of D ₂ -D ₁ ) is detected based on sequentially determining whether or not it exceeds each minute portion. Thereafter, the fluorescent particle discriminating means 9 compares the detection result of the skirt detecting means 8 with the reference value from the reference value generating means 15, and the measured fluorescent particles are specific fluorescent particles. It is determined whether or not.

For example, when the excitation light is irradiated to a mixture of white blood cells and impurities, the fluorescence intensity distribution of white blood cells has a shape as shown in FIG. 3 (a), for example. The distribution shows, for example, a shape in which the skirt portion is widened. In this case, the combined signal of white blood cells has the shape shown by z = g ₁ (x) in FIG. Of z = g ₂ (x). Therefore, even if the first threshold value C ₁ is the difference between the two small, the difference between them is the second threshold value C ₂ increases, the ability to accurately determine both.

Incidentally, the fluorescent particle discriminating means 9 performs discrimination based on the detection result of the skirt detecting means 8 (that is, the size of the distribution area of the skirt). The determination may be made by taking into account numerical values other than the result. For example, the size of the distribution region of the peak portion detected by the skirt detection unit 8 is detected by detecting the size of the distribution region of the peak portion (not only the position of the peak portion) by the peak detection means 7. Whether the fluorescent particle being measured is a specific fluorescent particle based on the size of the distribution region of the peak portion detected by the peak portion detecting means 7 and the reference value from the reference value generating means 15 The fluorescent particle discriminating means 9 may discriminate this. In such a case, the discrimination accuracy can be improved (as compared with the case where discrimination is made based only on the size of the distribution area of the skirt). Specifically, the size of the distribution region of the skirt portion and S _L, if the size of the distribution region of the peaks was set to S _H, the fluorescent particles discriminating means 9,
· S _L / _S ratio, the _{H (S L -S H) /} S may _H determined based on the value or the like of the ratio-of _(S L -S _H). In this case, the determination is performed using the two threshold values of the first threshold value and the second threshold value, but of course the present invention is not limited to this, and the determination may be performed using three or more threshold values. .

Incidentally, as illustrated in FIG. 3 (c), the second threshold value C _2, the first smaller than the threshold value C _1, and the A portion shown by the outer portion (symbol D ₃ valleys G ₁ The non-fluorescent part where no light is emitted by light excitation) is set to a value substantially equal to the value of the synthetic signal g (x, y) or g (x) (see symbol E). In general, the composite signal of leukocytes and the composite signal of contaminants are the second threshold value C ₂ (that is, the composite signal g (x, y) or g (x) in the outer portion of the valley portion G ₁ ). (Thresholds approximately equal to the values) greatly differ (see z = g ₁ (x) and z = g ₂ (x) in FIG. 4), so that white blood cells and contaminants can be clearly distinguished.

Incidentally, the peak portion detecting means 7 described above, as shown in FIG. 1, the first threshold value generator 70 for outputting a first threshold value C _1, the combined signal g (x), the value of g (x, y) There the first threshold value comparison means 71 for determining whether it exceeds the first threshold value C _1, it may be configured by. It is also possible to provide a first counting means 72 for counting the number of micro parts exceeds the first threshold value C _1. Skirt detecting means 8 described above be similar, the second threshold value generator 80 for outputting a second threshold value C _2, said combined signal g for each minute portion (x), the value of g (x, y) a second threshold comparison means 81 for determining whether it exceeds the second threshold value C _2, it may be configured by. It is also possible to provide a second counting means 82 for counting the number of minute portion exceeding the second threshold value C _2.

Note that the optimum value of the first threshold C ₁ is
An illumination state of the excitation light with respect to the fluorescent particles,
The type of subject,
・ Strength of action of surfactant and fluorescent dye (mainly determined by concentration),
Depends on etc. For example, the value of the low concentration of the fluorescent dye synthesized signal becomes totally low, should be lower accordingly first threshold value C ₁ is also. The case where the optimum value of the first threshold value C ₁ in accordance with the illumination condition of the excitation light is different,
・ If there is unevenness in the irradiation of the excitation light and the illumination state is different between one part and another part,
-When the illumination intensity of the excitation light changes with time, and the illumination state differs between one irradiation timing and another irradiation timing,
Etc. Therefore, the first threshold value generating means 70, a first threshold value C ₁ proper in accordance with the illumination condition such as good idea as to the operation output. For example, it arranged in such a way as uneven irradiation of the exciting light as described above (the locational unevenness) can be detected by the sensor, to change the first threshold value C ₁ spatially in accordance with the irradiation unevenness or, the irradiation intensity be prepared to detect a temporal change, it is advisable to be able to change the first threshold value C ₁ in accordance with the intensity change. Thereby, the fluorescent particles can be accurately determined even if the illumination state is not constant. In addition, since it is difficult to obtain | require such an optimal value by calculation, it is good to obtain | require by experiment. For example, several types of surfactants, fluorescent dyes, several types of the first threshold value C ₁ optimal with respect to the subject or the like may be experimentally obtained in advance and saves the value as table data, the first threshold value generating means 70 may keep the read the first threshold value C ₁ appropriately from the table data. The optimum value of the second threshold value C ₂ is also a good idea in the same manner.

Further, as described above, when performing discrimination of the fluorescent particles by using the area S _H of the distribution region of the peaks, the ratio S _{H /} S _L and the area S _L of the distribution region of the skirt portion, FIG. 1 to generate a reference value of _S H / _{S L} by the reference value generating means 15 shown, the fluorescent particles at _S H / _{S L} calculation unit 90 of the determination means 9 _S H / _{S L} value (calculated value) It is preferable to calculate and compare the appropriate value and the calculated value by the S _H / S _L comparing means 91.

Incidentally, the skirt portion detection unit 8 is adapted to detect the size of the distribution region of the skirt at or exceeds the second threshold value C _2, the portions other than the skirt portion and the second threshold value C ₂ In some cases (for example, refer to the part indicated by symbol E for the solid curve indicated by z = g ₁ (x) in FIG. 4), and even such a part is detected, the determination result is incorrect. There is a risk that. In addition, when the fluorescent particle determination unit 9 performs the fluorescent particle determination in consideration of the size of the distribution region of the peak portion in addition to the size of the distribution region of the peak portion, the skirt used for the determination is used. The size of the distribution region of the part and the size of the distribution region of the peak part must be for the same fluorescent particle, the size of the distribution region of the tail part for one fluorescent particle and the peak for another fluorescent particle If the determination is made based on the size of the distribution area of the part, the determination result may be wrong. In contrast, in the present invention, whether it exceeds a second threshold value discrimination, from the side close to the peak portion only takes place for the part up to the side close to the valley G _1, the valley portion since about the outer portion D ₃ not performed, you can avoid the influence of noise outside of the valley portions (or prevent you from detecting the size of the distribution region of the skirt portion for another fluorescent particles Yes, it is possible to accurately identify fluorescent particles. For example, when white blood cells (fluorescent particles) and foreign matters (cell components other than white blood cells and dust and dust) are contained in the fluorescent particle containing means 2, white blood cells and foreign matters can be accurately discriminated.

It should be noted that at least one memory means (see reference numeral 13 in FIG. 5) for storing the value of the synthesized signal for a predetermined region including the peak portion is provided, and the skirt detecting means 8 is the memory means. 13 combined signal stored in the g (x, y) or g if the value of (x) exceeds the second threshold value C ₂ to sequentially determine a minute each portion from the side close to the peak portion based, for detecting the size of the distribution region of the skirt exceeding the second threshold value C _2, may be so. Thereby, the discrimination time of fluorescent particles can be shortened and the discrimination accuracy can be improved. This will be specifically described below.

That is, first, the extracts of the minute portion exceeding the first threshold value C ₁ performed by the peak portion detecting means 7 as described above (see S1 in FIG. 6). Then, if seven minute parts indicated by “(1)” in FIG. 7 are extracted, labeling information (address information) of minute parts (parts not yet extracted) adjacent to the upper, lower, left, and right of each minute part a synthesized signal value (value of the composite signal at each minute portion) is stored in the memory unit 13 (see S2 in Figure 6), the minute portion exceeding the second threshold value C ₂ based on the stored combined signal values Extraction is performed by the skirt detection means 8 (see S3 in FIG. 6). Now, the minute portion indicated by "(2)" in FIG. 7 is greater than the second threshold value C _2. Then, the labeling information and the synthesized signal value are stored in the memory means 13 (see S4 and S2 in FIG. 6) for the minute parts (parts not yet extracted; hereinafter the same) adjacent to the minute parts. ) extracts a small portion exceeding the second threshold value C ₂ on the basis of the stored combined signal value by the skirt portion detecting means 8 (see S3 in FIG. 6). Now, the minute portion indicated by "(3)" in FIG. 7 exceeds the second threshold value C _2, a minute portion indicated by "× 3" does not exceed the second threshold value C _2. Then, the labeling information and the synthesized signal value are stored in the memory means 13 (see S4 and S2 in FIG. 6) for the minute part adjacent to the minute part indicated by “(3)” in the vertical and horizontal directions, and the stored synthesized signal is stored. to extract minute portion exceeding the second threshold value C ₂ based on the value by skirt detecting means 8 (see S3 in FIG. 6). Note that a determination is not made for a minute portion indicated by “−” (that is, a minute portion adjacent to the minute portion indicated by “× 3”). Hereinafter, the same determination is made. The minute portion exceeding the second threshold value C ₂ is, "(4)""(5)""(6)""(7)""(8)""(9) in order as shown in" go extracted stand in order as indicated by the second threshold value _{C 2} of the minute portion is "× 4" does not exceed "× 5""×6""×7""×8""×9""×10" I will do it. The portions indicated by “× 3” to “× 10” are portions corresponding to “valley portions”. Then, when it exceeds a predetermined area (in FIG. 7, an area composed of 121 minute portions of 11 rows × 11 columns), the determination is stopped (see S4 and S5 in FIG. 6). Since the fluorescent particles can be identified only by comparison within the area, there is no need to widen the area.

Here, in the apparatus shown in FIG. 5, only one memory means 13 is provided.
Storing the combined signal value and labeling information of each minute portion output from the adding means 6 in the first memory means;
-Store the combined signal value and labeling information of the minute part indicated by reference numeral "(2)" in FIG. 7 in the second memory means,
-Store the combined signal value and labeling information of the minute part indicated by reference numeral "(3)" in FIG. 7 in the third memory means,
-Stores the combined signal value and labeling information of the minute part indicated by reference numeral "(4)" in FIG. 7 in the fourth memory means,
It may be said that.

    In addition, you may measure a fluorescent particle by the method shown in FIG.

First, the first counting means 72 described above, for each fluorescent particles, and calculates and storage area S _H of the portion exceeding the first threshold value C ₁ (see S11). Next, for each fluorescent particle, information (synthetic signal value and labeling information) about the minute portion around the fluorescent particle is stored in the memory means (see S12). Based on the stored information, the second counting means 82 described above, for each fluorescent particles, to calculate and store the area S _L of the portion exceeding the second threshold value C ₂ (see S13). Thereafter, the _S H / _{S L} calculation unit 90, for each fluorescent _particles, calculates a value of _S H / S _L (see S14). Wherein _S H / _{S L} comparator _91, (see S15) _S H / S _L of comparing the calculated value of the reference values R and _S H / _{S L,} predetermined fluorescence in the case toward the calculation value is smaller If it is a particle, it is counted (see S16), and if the reference value is smaller, it is not counted as a predetermined fluorescent particle (see S17). This calculation and comparison are sequentially performed for each fluorescent particle (see S18, S15, S16, and S17). And discrimination | determination is complete | finished about all the fluorescent particles, and a count is stopped (refer S18 of FIG. 8, S19).

    By the way, the excitation light source 3 described above needs to be selected in accordance with the light absorption characteristics of the fluorescent dye (dye for fluorescently staining leukocytes or the like). For example, when propidium iodide (PI) is used as the fluorescent dye, a light source that generates excitation light (green light) in a wavelength region of about 500 to 550 nm is preferable. Furthermore, in order to obtain a fluorescence intensity sufficient for detection, the excitation light source also needs to have a sufficient output. Specifically, a green laser, a semiconductor laser-induced solid-state laser, a green LED array, a high-intensity LED, or the like can be used. Note that a light projecting system 11 for adjusting the degree of concentration of excitation light and the like may be disposed between the excitation light source 3 and the fluorescent particle storage means 2.

    As the fluorescence measuring means 4, a CCD camera is the simplest in configuration, but a two-dimensional photodetector such as a CMOS camera, a line sensor, a photodetector, a photodetector array, and a mechanism for moving an observation point; It can also be realized in combination.

    Further, a filter means 12 for allowing only the light generated by the fluorescence to pass (not allowing light in other wavelength ranges to pass) is arranged between the fluorescent particle accommodating means 2 and the fluorescence measuring means 4. good. For example, when PI is used as the fluorescent dye, a filter that cuts off the used excitation light (for example, light of 550 nm or less) and transmits light of around 600 nm is used. As the type of filter, a filter capable of controlling spectral characteristics, such as a colored glass filter and a dielectric multiphase film filter, can be used.

A fluorescent particle counting device (see reference numeral 10 in FIG. 1) for counting the number of specific fluorescent particles based on the determination result of the fluorescent particle determining device 9 is connected to the fluorescent particle determining device 1, and the fluorescent particle counting device May be configured. This according to the fluorescent particle counting device, whether it exceeds a second threshold determination is only performed for the part up to the side close to the valley G ₁ from the side adjacent to the peak portion, the valley portion since about the outer portion D ₃ not performed, can avoid the influence of noise outside of the valley portion, it is possible to accurately perform the counting of fluorescent particles. For example, when white blood cells (fluorescent particles) and impurities (cell components other than white blood cells, dust and dust) are stored in the fluorescent particle storage means 2, only white blood cells can be accurately counted.

    The fluorescent particle discrimination device according to the present invention may be configured as shown in FIGS.

    That is, it is preferable to connect a signal converter 14 to the fluorescence measuring means 4 described above so as to convert an analog signal from the fluorescence measuring means 4 into a digital signal. In the case where a CCD camera is used as the fluorescence measuring means 4, a video capture board incorporating an A / D conversion circuit may be used as the signal converter 14. Further, when a combination of a line sensor or photodetector and a mechanism for moving the observation point is used as the fluorescence measuring means 4, a circuit for reconstructing the signal obtained by the signal converter 14 as an image is provided. It is necessary to provide it.

    Further, a personal computer 20 is connected to the signal converter 14, and the above-described differentiation means 5, addition means 6, peak detection means 7, skirt detection means 8 and fluorescent particle discrimination based on predetermined software in the personal computer. Means 9 and the like may be provided.

    It is preferable that various information can be displayed on the monitor 21 of the personal computer 20.

Furthermore, it is preferable to provide an arithmetic processing unit 200 in the personal computer as shown in FIG. 10 and perform noise removal by performing smoothing processing or Laplacian filter processing of the captured image data. In addition, a plurality of image memories M ₁ , M ₂ ,... And an image data transfer control unit 201 may be provided in the personal computer, and image data may be transferred as appropriate.

For example, the captured image data is transferred to the image memory M ₁ by the image data transfer control unit 201. The transferred image data is subjected to image processing by the arithmetic processing unit 200, and noise that hinders subsequent image analysis is removed. First, a smoothing process to the stored image data in the image memory M ₁ addition, the original image data remains stored in the image memory M _1, to build the image after the smoothing process to the image memory M ₂ storage. Thereafter, Laplacian filtering process on the image data after stored smoothed process in the image memory M _2, stores the processed image in the image memory M _3.

    Next, a method for discriminating fluorescent particles according to the present invention will be described.

The method for discriminating fluorescent particles according to the present invention includes the following steps. That is,
A step of irradiating the fluorescent particles with excitation light, a step of capturing fluorescence generated based on the irradiation of the excitation light and measuring its intensity distribution, and intensity distribution signals f (x, y) and f (f) obtained by the measurement x) and differentiating signals f ′ (x, y) and f ′ (x) by outputting the differential signals f ′ (x, y) and f ′ (x). y) and f ′ (x) are added to output the combined signals g (x, y) and g (x). The value of the combined signal g (x, y) g (x) is the first threshold value. wherein the side close to the step-the peak portion for detecting the peak portion exceeding the first threshold value C ₁ basis to determine if exceeds (code reference C ₁ in FIG. 3 (c)) valleys to the side close to the part G _1, the value of the combined signal g (x, y) or g (x) exceeds the second threshold value (reference symbol C ₂ in FIG. 3 (c)) Detection result of whether the size of the distribution region of the step-該裾unit for detecting the size of the distribution region of the skirt exceeding the second threshold value C ₂ based on the sequentially determined for each minute portion Determining whether or not the fluorescent particle being measured is a specific fluorescent particle based on the comparison with the reference value

    Note that the number of fluorescent particles may be counted after the above-described determination of the fluorescent particles.

    By the way, the method for discriminating fluorescent particles according to the present invention can discriminate white blood cells in blood, platelet preparations or red blood cell preparations, or perform white blood cell counting. In addition, it is necessary to dissolve the leukocyte cell membrane and fluorescently stain the leukocyte cell nucleus (S22 in FIG. 13).

    In addition, this inventor has confirmed the effect of this invention with the following method.

    First, a platelet preparation-like solution in which a known number of leukocytes were mixed was diluted to prepare a plurality of specimens having different leukocyte concentrations. Then, when the number of white blood cells in each subject was measured with the conventional apparatus and the apparatus according to the present invention, the measurement result of the conventional apparatus is as shown by “□” in FIG. 11 (a), and the apparatus according to the present invention. The measurement results were as shown in “●” in the figure. Similarly, a red blood cell preparation-like solution mixed with a known number of white blood cells was diluted to prepare a plurality of types of specimens having different white blood cell concentrations. Then, when the number of white blood cells in each subject was measured with the conventional apparatus and the apparatus according to the present invention, the measurement result of the conventional apparatus is as shown by “□” in FIG. 11 (b), and the apparatus according to the present invention. The measurement results were as shown in “●” in the figure. If the various solutions are diluted, the concentration of leukocytes should change as shown by the one-dot chain line in each figure, but the measurement result of the device according to the present invention is the one-dot chain line rather than the measurement result of the conventional device. You can see that The regression line of both data is obtained by calculation and the table is shown in FIG. 11 (c). From this table, the measurement result of the present invention has improved both the slope of the regression line (ideally closer to 1) and the y-intercept (ideally closer to 0) than the conventional method. It can be seen that highly accurate measurement was performed.

FIG. 1 is a block diagram showing an example of the structure of a fluorescent particle discriminating apparatus and a fluorescent particle counter according to the present invention. 2A is a diagram showing an example of the fluorescence intensity distribution (two-dimensional distribution) of leukocytes, and FIG. 2B is a diagram showing an example of the fluorescence intensity distribution (two-dimensional distribution) of impurities. . Fig.3 (a) is a figure which shows an example of the fluorescence intensity one-dimensional distribution (intensity distribution signal) of a fluorescent particle, FIG.3 (b) shows the differential signal which differentiated the intensity distribution signal of the figure (a). FIG. 3 (c) is a diagram showing a combined signal obtained by adding the differential signal shown in FIG. 3 (b) to the intensity distribution signal shown in FIG. 3 (a). FIG. 4 is a waveform diagram for explaining the difference between the synthetic signal of leukocytes and the synthetic signal of impurities. FIG. 5 is a block diagram showing another example of the structure of the fluorescent particle discriminating apparatus and the fluorescent particle counting apparatus according to the present invention. FIG. 6 is a flowchart for explaining a method of discriminating fluorescent particles. FIG. 7 is a schematic diagram for explaining a fluorescent particle discrimination method. FIG. 8 is a flowchart for explaining an example of a fluorescent particle counting method. FIG. 9 is a block diagram showing an example of the structure of the fluorescent particle discrimination device according to the present invention. FIG. 10 is a block diagram for explaining the detailed structure thereof. FIG. 11 is a diagram for explaining the effect of the present invention. FIG. 12 is a block diagram showing an example of the structure of a conventional apparatus for counting white blood cells. FIG. 13 is a flowchart showing an example of a white blood cell counting method. FIG. 14 is a diagram for explaining white blood cell fluorescence image signals, foreign matter fluorescence image signals, and conventional problems.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Fluorescent particle discrimination | determination apparatus 2 Fluorescent particle accommodation means 3 Excitation light source 4 Fluorescence measurement means 5 Differentiation means 6 Addition means 7 Peak part detection means 8 Bottom part detection means (rising part detection means)
DESCRIPTION OF SYMBOLS 9 Fluorescent particle discrimination | determination means 10 Fluorescent particle count means 13 Memory means 70 1st threshold value generation means 71 1st threshold value comparison means C ₁ 1st threshold value C ₂ 2nd threshold value f (x, y), f (x) Fluorescence intensity distribution Intensity distribution signal shown f ′ (x, y), f ′ (x) Differential signal g (x, y), g (x) Composite signal

Claims (7)

Fluorescent particles comprising an excitation light source for irradiating the stored fluorescent particles with excitation light, and fluorescence measurement means for capturing fluorescence generated by the excitation light irradiation and measuring its intensity distribution In the discrimination device,
Differential means for outputting a differential signal obtained by differentiating the intensity distribution signal from the fluorescence measurement means;
Based on the addition of the differential signal to the intensity distribution signal, a combined signal having a peak shape including a peak portion and a rising portion connected to the peak portion and having a valley portion outside the rising portion is output. Adding means;
Peak part detecting means for detecting the peak part exceeding the first threshold based on determining whether the value of the combined signal exceeds the first threshold;
Based on sequentially determining for each minute portion whether the value of the combined signal exceeds the second threshold value from the side close to the peak portion detected by the peak portion detecting means to the side close to the valley portion. Rising portion detecting means for detecting the size of the distribution region of the rising portion exceeding the second threshold;
A reference value generating means for generating a reference value serving as a reference when discriminating whether the particle is a specific fluorescent particle;
A fluorescent particle discriminating unit that discriminates whether or not the fluorescent particle being measured is a specific fluorescent particle based on the detection result of the rising portion detecting unit and the reference value from the reference value generating unit;
A fluorescent particle discriminating apparatus comprising The peak part detection means detects the size of the distribution region of the peak part,
The fluorescent particle discriminating means includes a size of the rising area distribution area detected by the rising edge detecting means, a size of the peak area distribution area detected by the peak detecting means, and a reference value generating means. Based on the reference value, determine whether the fluorescent particle being measured is a specific fluorescent particle,
The fluorescent particle discriminating apparatus according to claim 1. The second threshold value is smaller than the first threshold value and substantially equal to the value of the combined signal in the outer portion of the valley;
The fluorescent particle discriminating apparatus according to claim 1 or 2. The peak portion detection means is composed of first threshold value generation means for outputting a first threshold value, and first threshold value comparison means for determining whether or not the value of the combined signal exceeds the first threshold value,
The first threshold generation means calculates and outputs the first threshold according to the illumination state of the excitation light with respect to the fluorescent particles.
The fluorescent particle discriminating apparatus according to any one of claims 1 to 3, wherein: Comprising at least one memory means for storing a value of the synthesized signal for a predetermined region including the peak portion;
The rising portion detecting means sequentially determines whether the value of the synthesized signal stored in the memory means exceeds a second threshold value for each minute portion from the side close to the peak portion. Detecting the size of the distribution region of the rising portion exceeding the threshold;
The fluorescent particle discrimination device according to any one of claims 1 to 4, wherein the fluorescent particle discrimination device. The fluorescent particle discrimination device according to any one of claims 1 to 5,
A fluorescent particle counting means for counting the number of specific fluorescent particles based on the determination result by the fluorescent particle determining device;
A fluorescent particle counting apparatus configured by providing Irradiating fluorescent particles with excitation light;
Capturing fluorescence generated based on irradiation of the excitation light and measuring its intensity distribution;
Differentiating the intensity distribution signal obtained by the measurement and outputting a differential signal;
Based on the addition of the differential signal to the intensity distribution signal, a combined signal having a peak shape and a rising portion connected to the peak portion and having a valley portion outside the rising portion is output. Process,
Detecting the peak portion exceeding the first threshold based on determining whether the value of the combined signal exceeds the first threshold; and
From the side close to the peak part to the side close to the valley part, the composite signal exceeds the second threshold based on sequentially determining whether the value of the composite signal exceeds the second threshold value for each minute part. Detecting the size of the distribution region of the rising portion;
Determining whether or not the fluorescent particles being measured are specific fluorescent particles based on the detection result and the reference value for the size of the distribution region of the rising portion; and
A method for distinguishing fluorescent particles comprising:

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