Particle analyzer

Description: TECHNICAL FIELD [Detailed description of the invention]

【0001】

[Technical field to which the invention belongs]

The present invention relates to a particle analyzer, and in particular, creates a two-dimensional frequency distribution map (scattergram) using particle characteristic parameters, fractionates a group of particles appearing in the distribution map, and specifies the type and number of particles. Regarding analyzers.

【0002】

[Conventional technology]

Conventionally, in this type of particle analyzer, a plurality of regions are set in advance on the distribution map, the degree of attribution of each of the plurality of particles appearing in the distribution map to the set region is calculated, and the fractionation region is determined according to the degree of attribution. Is known (see, for example, Japanese Patent Application Laid-Open No. 6-3252).

【0003】

[Problems to be Solved by the Invention]

By the way, when a conventional particle analyzer is used as a blood analyzer that analyzes blood cells in blood, for example, an element that detects electrical or optical information from the blood cells, the type and amount of a reagent that dilutes the blood to be measured, and the blood cells. When the feature parameters used to create the distribution map fluctuate due to some cause, such as dirt in the blood cells, changes in the amplification degree of the electric circuit that converts the information into electrical signals in order to obtain feature parameters from the detected information, etc. Has a problem that a group of particles appearing in a two-dimensional frequency distribution map moves and correct fractionation is not performed, resulting in an erroneous analysis result.

【0004】

The present invention has been made in consideration of such circumstances, and provides a particle analyzer having a function of determining an erroneous fractionation as a fractionation abnormality when an erroneous fractionation is performed.

【0005】

[Means for solving problems]

The present invention includes a detection unit that detects feature parameters from each of a plurality of particles, a distribution map creation unit that creates at least two two-dimensional frequency distribution maps using the detected feature parameters, and particles that appear in each distribution map. A fractionation unit that fractionates particles into particle groups, and a calculation unit that compares the number of particles in those particle groups when the particle groups containing particles of the same type in the two distribution maps are fractionated. Provided is a particle analyzer provided with a determination unit for determining an abnormality in fractionation in the distribution map based on a comparison result.

【0006】

BEST MODE FOR CARRYING OUT THE INVENTION

The target particles of the present invention are tangible substances mainly contained in body fluids such as blood and urine, but may be particles made of industrial inorganic or organic substances. For the detection unit of the present invention, for example, a flow cytometer, that is, a device including a flow cell in which a particle-containing liquid is wrapped in a sheath liquid and flowed, and an optical element for detecting characteristic parameters from each particle of the particle-containing liquid can be used. .. In this case, the detected feature parameters include optical information based on forward scattered light, laterally scattered light, fluorescence (for example, lateral fluorescence), and the like. In the flow cytometer, these various optical informations are photoelectrically converted by the optical element to obtain a pulse signal according to the characteristics of the particles, but the peak level is used as the light intensity or the pulse signal exceeds a predetermined threshold value. The time spent can be used as a feature parameter by using the pulse width as the pulse width. That is, the forward scattered light information includes the forward scattered light intensity and the forward scattered light pulse width, the lateral scattered light information includes the lateral scattered light intensity and the lateral scattered light pulse width, and the lateral fluorescence information includes the lateral fluorescence. Intensity and lateral fluorescence pulse width can be the feature parameters.

【0007】

In addition, as the two-dimensional frequency distribution map created by the distribution map creation unit, when a flow cytometer is used as the detection unit, for example, a distribution map with lateral scattered light intensity and lateral fluorescence intensity as parameters, lateral A distribution map with scattered light intensity and forward scattered light intensity as parameters, a distribution map with lateral fluorescence intensity and forward scattered light intensity as parameters, and a distribution map with lateral fluorescence intensity and forward scattered light intensity as parameters are listed. Be done.

【0008】

As a fractionation method in which the fractionation section fractionates the particles of the distribution map into a group, a conventionally known method, for example, the method described in JP-A-6-3252 can be used.

【0009】

Further, the fractionation unit, the calculation unit, and the determination unit in the present invention can be integrally configured by a microcomputer or a personal computer composed of a CPU, ROM, and RAM.

【0010】

In addition, when the detection unit consists of a flow cytometer and the feature parameters are forward scattered light intensity, side scattered light intensity and lateral fluorescence intensity, the two-dimensional frequency distribution map parameters the lateral fluorescence intensity and forward scattered light intensity. A first distribution map and a second distribution map in which the lateral scattered light intensity and the lateral fluorescence intensity are used as parameters may be used.

【0011】

In this case, if the detected particles are blood cells, the fractionation section fractions leukocytes (neutrophils, basophils, eosinophils, lymphocytes, monocytes) in the first distribution map and distributes the second. In the figure, neutrophils, basophils and eosinophils as components of white blood cells can be fractionated.

【0012】

At this time, the calculation unit determines the number N of leukocytes (neutrophils, basophils, eosinophils, lymphocytes, monocytes) in the first distribution map, and neutrophils, basophils, and eosinophils in the second distribution map. The sum M of the number of spheres is calculated and compared with M and N, and the determination unit can determine that the fractionation of the first distribution map is abnormal when N <M.

【0013】

Further, the present invention comprises a quantification unit for quantifying a particle-containing sample, a sample preparation unit for preparing first and second samples using the quantified sample, and the adjusted first sample and second sample. A detection unit that measures each sample and detects a plurality of feature parameters from the particles in each sample, a distribution map creation unit that creates a two-dimensional frequency distribution map based on the detected feature parameters for each sample, and each distribution. The fractionation part that fractionates the particles appearing in the figure into particle groups, and the particle group that contains particles of the same type in the distribution map created for the first sample and the distribution map created for the second sample are separated. Provided is a particle analyzer provided with a calculation unit that compares the number of particles of those particle groups when a sample is drawn, and a determination unit that determines an abnormality in the fractionation in the distribution map based on the comparison result by the calculation unit. To do. Example Hereinafter, the present invention will be described in detail based on the examples shown in the drawings. A common number and a symbol are added to the common elements of each drawing.

【0014】

Configuration of blood analyzer FIG. 1 is a perspective view showing an optical system of a blood analyzer using the method of the present invention. In the figure, the beam emitted from the laser diode 21 irradiates the orifice portion 13 of the sheath flow cell 1 via the collimating lens 22. The forward scattered light emitted from the blood cells passing through the orifice portion enters the photodiode 26 via the condenser lens 24 and the pinhole plate 25.

【0015】

On the other hand, regarding the laterally scattered light and the lateral fluorescence emitted from the blood cells passing through the orifice portion 13, the laterally scattered light is passed through the condensing lens 27 and the dichroic mirror 28 and is referred to as a photomultiplier tube (hereinafter referred to as photomal). ) 29, and the lateral fluorescence is incident on Photomal 31 via the condenser lens 27, the dichroic mirror 28, the filter 36, and the pinhole plate 30.

【0016】

The forward scattered light signal output from the photodiode 26, the side scattered light signal output from the photomultiplier 29, and the side fluorescence signal output from the photomultiplier 31 are amplified by the amplifiers 32, 33, and 34, respectively. Is input to the analysis unit 35.

【0017】

FIG. 2 is a system diagram showing the fluid system of the blood analyzer shown in FIG. In the figure, first, in the cleaning step, valves 41 and 50 are opened, and the sheath liquid is sent from the sheath liquid chamber 42 containing the sheath liquid by the pressure P applied from the pressure device 43, and the valve 41 and the metering syringe 44 are sent. It is discharged to the waste liquid chamber 45 through the nozzle 6 and the waste liquid chamber 45 through the valve 50 and the sheath flow cell 1, and the valves 41 and 50 are closed after a predetermined time. As a result, the metering syringe 44, the nozzle 6, the sheath flow cell 1 and its path are washed with the sheath liquid.

【0018】

Next, in the measurement step, the valves 46 and 47 are opened, and the sample liquid is sucked by the negative pressure of the suction device 49 from the reaction chamber 48 containing the blood-containing sample liquid by reacting with the reagent, and the valves 46 and the nozzle 6 are sucked. Valves 46 and 47 are closed when the path between them is filled with sample fluid. Next, when the valve 50 is opened, the sheath liquid is sent from the sheath liquid chamber 42 to the sheath flow cell 1 by the pressure of the pressure device 43, and is discharged to the waste liquid chamber 45.

【0019】

Next, when the valve 41 is opened, the pressure P from the pressure device 43 is also transmitted to the tip of the nozzle 6 via the metering syringe 44, and the pressure of the sheath liquid outside the nozzle and the sample inside the nozzle at the tip of the nozzle 6 Equilibrate with the pressure of the liquid. Therefore, when the piston 44b of the metering syringe 44 is driven by the motor 44a in this state, the sample liquid existing between the valve 46 and the nozzle 6 is easily discharged from the nozzle 6 to the orifice portion 13, and is finely squeezed by the sheath liquid. It passes through the orifice portion 13 and is discharged to the waste liquid chamber 45 together with the sheath liquid. Then, when the driving of the piston 44b of the metering syringe 44 is completed, the measurement process is completed.

【0020】

Next, the motor 44a reverses and the piston 44b is pulled back, and the metering syringe 44 returns to the initial state, but since the valves 41 and 50 remain open during this period, the above-mentioned cleaning step is performed, and then the cleaning step is performed. Will be prepared for the measurement process of.

【0021】

Therefore, the measurement can be performed on the other sample liquids contained in the other reaction chambers 51, 52, 53 by opening and closing the valves 54, 55, 56 and sequentially executing the same steps as described above. The valve 57 is a valve for discharging the waste liquid from the waste liquid chamber 45, and is opened and closed as needed.

【0022】

FIG. 3 is a block diagram showing the configuration of the analysis unit 35 of FIG. In FIG. 3, 61 is a data input unit for presetting conditions such as various numerical values and areas, and is composed of, for example, a keyboard and a mouse.

【0023】

Further, 62 is a setting condition storage unit for storing various set conditions, and 63 is a data storage unit for storing optical information obtained from the output signals of the photodiode 26 and the photomultiplier tubes 29 and 31. 64 is two-dimensional using the optical information stored in the data storage unit 63, that is, one of two parameters: forward scattered light intensity (Fsc), side scattered light intensity (Ssc), and side fluorescence intensity (Sfl). The distribution map creation unit that creates the frequency distribution map, and 65 is the extraction unit that extracts coordinates and regions from the distribution map created by the distribution map creation unit 64.

【0024】

66 is a fractionation area determination unit that determines the fractionation area of each particle in the distribution map created by the distribution map creation unit 64, and 67 is a calculation unit that counts the number of particles in the fractionation area and compares the counting results. Reference numeral 70 denotes a determination unit for determining an abnormality in the fractionation in the distribution map based on the comparison result. Then, the calculation result of the calculation unit 67 and the judgment result of the judgment unit 70 are displayed on the display unit 68 together with the distribution map created by the distribution map creation unit 64. Reference numeral 69 denotes a fluid system drive unit for driving the valves 41,46,47,50,54,55,56,57 and the motor 44a shown in FIG. The analysis unit 35 is composed of a personal computer.

【0025】

Creating a two-dimensional frequency distribution map FIG. 8 shows a sample, a quantification unit, and a reagent supply unit, which are not shown in FIG. 2, and is a diagram for explaining in detail the preparation of the sample. As shown in FIG. 8, blood (sample) from the sample container 80 is aspirated and quantified by the quantification units 81 to 84 in the required amount, and distributed to the reaction chambers 48, 51, 52, and 53, respectively. That is, the quantified blood for measurement in the "nucleated red blood cell measurement mode" is distributed to the reaction chamber 48. The quantified blood for measurement in the "white blood cell, basophil measurement mode" is distributed to the reaction chamber 51. The quantified blood for measurement in the "white blood cell classification measurement mode" is distributed to the reaction chamber 52. The quantified blood for measurement in "reticulocyte measurement mode" is distributed to reaction chamber 53. Then, a predetermined reagent is supplied to each of the reaction chambers 48, 51, 52, and 53 by the corresponding reagent supply units 85 to 88, and the blood reacts with the reagent. In this way, a plurality of samples corresponding to each measurement mode are prepared from one sample and sequentially measured in the sheath flow cell 1. That is, in the input unit 61 (Fig. 3), there are four measurement modes: "nucleated erythrocyte measurement mode", "white blood cell and basophil measurement mode", "white blood cell classification measurement mode", and "reticulocyte measurement mode". When each order is set, each measurement mode is executed as follows according to the order.

【0026】

Nucleated red blood cell measurement mode In the "nucleated red blood cell measurement mode", 18 μl of blood is carried to the reaction chamber 48 together with 882 μl of Stomatolyzer NR hemolytic agent (manufactured by Sysmex Corporation). Then, 18 μl of Stomatolyzer NR stain (manufactured by Sysmex Corporation) is added. By reacting in this state for about 7 seconds, erythrocytes are hemolyzed and leukocytes / nucleated erythrocytes are stained.

【0027】

The sample subjected to this treatment is ejected from the nozzle 6 by the metering syringe 44, and among the information obtained by optical measurement, the lateral fluorescence intensity (Sfl) and the forward scattered light intensity (Fsc) are two-dimensional. Figure 4 shows an example of the frequency distribution map. In FIG. 4, the nucleated red blood cell and leukocyte populations are fractionated, respectively.

【0028】

White blood cell, basophil measurement mode In the "white blood cell and basophil measurement mode", 18 μl of blood is carried to the reaction chamber 51 together with 882 μl of Stomalyzer FB (II) (manufactured by Sysmex Corporation). By reacting in this state for about 14 seconds, erythrocytes are hemolyzed, and leukocytes other than basophils are naked nucleated and contracted.

【0029】

The sample subjected to this treatment is ejected from the nozzle 6 by the metering syringe 44, and among the information obtained by optical measurement, the lateral scattered light intensity (Ssc) and the forward scattered light intensity (Fsc) are calculated as 2. Figure 5 shows an example of a dimensional frequency distribution map. In FIG. 5, groups of basophils and (lymphocytes + monocytes + neutrophils + eosinophils) are fractionated, respectively.

【0030】

White blood cell classification measurement mode In the "white blood cell classification measurement mode", 18 μl of blood is carried to the reaction chamber 52 together with 882 μl of Stomalyzer 4DL (manufactured by Sysmex Corporation). Then, 18 μl of Stomalyzer 4DS (manufactured by Sysmex Corporation) is added. By reacting in this state for about 22 seconds, red blood cells are hemolyzed and white blood cells are stained.

【0031】

The sample subjected to this treatment is ejected from the nozzle 6 by the metering syringe 44, and among the information obtained by optically measuring, the lateral scattered light intensity (Ssc) and the lateral fluorescence intensity (Sfl) are calculated as 2. Figure 6 shows an example of a dimensional frequency distribution map. In FIG. 6, populations of lymphocytes, monocytes, neutrophils + basophils, and eosinophils are fractionated, respectively.

【0032】

Reticulocyte measurement mode In the "reticulocyte measurement mode", 4.5 μl of blood is delivered to the reaction chamber 53 together with 895.5 μl of Letsearch (II) diluent (manufactured by Sysmex Corporation). Then, 18 μl of Let Search (II) staining solution (manufactured by Sysmex Corporation) is added. By reacting in this state for 31 seconds, reticulocytes and the like are stained.

【0033】

The sample subjected to this treatment is ejected from the nozzle 6 by the metering syringe 44, and among the information obtained by optical measurement, the lateral fluorescence intensity (Sfl) and the forward scattered light intensity (Fsc) are two-dimensional. Figure 7 shows an example of the frequency distribution map. In FIG. 7, reticulocytes, mature erythrocytes, and platelet populations are fractionated, respectively. FIG. 9 is a flowchart showing the operation of the blood analyzer of the present invention. Using this flowchart, a series of steps of the above sample preparation and measurement steps will be organized and described. Step S1: Aspirate the blood drawn from the patient. Step S2: Quantify and distribute the amount required for measurement in each measurement mode. Steps S3a to S3d: To each quantified blood, a predetermined reagent such as a diluting solution, a staining solution, or a hemolytic agent is added according to the measurement mode to perform a reaction treatment, and each measurement mode (nucleated white blood cell measurement mode, Prepare a sample for each white blood cell and basophil measurement mode, "white blood cell 4 classification measurement mode", and "reticulocyte measurement mode"). Steps S4a to S4d: Each sample prepared for each measurement mode is sequentially sent to the detection unit, and the detection unit detects optical information. Steps S5a to S5d: Create a two-dimensional frequency distribution map for each measurement mode based on the detected optical liquid information. Steps S6a to S6d: On each created distribution map, the particles that appear are fractionated and counted for each particle type. Step S7: Determine the presence or absence of fractionation abnormality based on the results of fractionation and counting in multiple distribution maps (this step is described in detail below).

【0034】

Judgment of fractionation abnormality In this example, "nucleated red blood cell measurement mode", "white blood cell and basophil measurement mode", and "leukocyte 4 classification measurement mode" are executed for one sample, and the distribution maps shown in FIGS. 4 to 6 are obtained. Then, the calculation unit 67 and the determination unit 70 (FIG. 3) determine the fractionation abnormality by the following procedure.

【0035】

First, the number N1 of white blood cells and the number N2 of nucleated red blood cells are calculated from the distribution diagram of the nucleated red blood cell measurement mode shown in FIG. 4, and it is determined whether or not the following equation holds. 100 × N1 / (N1 + N2) <10 ...... (1) [0036]

If equation (1) holds, it indicates that the number of nucleated red blood cells N2 is abnormally high relative to the number of white blood cells N1, which means that the sample was taken from an unhealthy patient or the distribution in FIG. In the figure, it means that the leukocyte is a fractionation abnormality that is mistakenly fractionated as nucleated red blood cell. That is, it is not possible to determine the fractionation abnormality in the "nucleated red blood cell measurement mode" only by this determination.

【0037】

Therefore, the fractionation abnormality is determined using the "nucleated red blood cell measurement mode" and the "white blood cell 4 classification measurement mode". In a sample in which nucleated red blood cells are present, nucleated red blood cells appear in the lymphocytes and the region below them in the distribution diagram of the leukocyte 4 classification measurement mode in FIG. The sum N3 of the number of nucleated red blood cells (neutrophils + basophils) and the number of eosinophils fractionated away from that region was counted, and the white blood cell count N1 obtained from FIG. Compare.

【0038】

And N3> N1 ...... (2) If so, the number N1 of white blood cells appearing in Fig. 4 will be less than the number N3 of some components of white blood cells appearing in Fig. 6, that is, (neutrophils + basophils + eosinophils), which causes a contradiction. Judge that the fraction is abnormal. That is, in this case, equation (2) is a condition indicating that most of the leukocytes were erroneously fractionated as nucleated red blood cells in FIG.

【0039】

When the number N3 of (neutrophil + basophil + eosinophil) is considerably smaller than the white blood cell count and the nucleated red blood cell count, the reliability of the above determination formula is low. In the case of a sample containing a large amount of nucleated red blood cells, the nucleated red blood cells are the basophils and (lymphocyte + monocyte + neutrophil + eosinophil) in the distribution map of "white blood cell, basophil measurement mode" in Fig. 5. Appears in the area. In the distribution map of Fig. 5, the number of particles N4 in the region of basophils and (lymphocytes + monocytes + neutrophils + eosinophils) that may contain nucleated erythrocytes was counted, and the leukocyte 4 classification in Fig. 6 was counted. Compare the number N3 of (neutrophils + basophils + eosinophils) shown in the distribution map of the measurement mode with the following formula. 100 × N3 / N4> 10 ...... (3) If the equation (3) is not satisfied, the determination of the equation (2) is not performed.

【0040】

In this way, when the determination unit 70 determines that the fraction in the distribution diagram of the nucleated red blood cell measurement mode of FIG. 4 is abnormal, the determination result is displayed on the display unit 68.

【0041】

[Effect of the invention]

According to the present invention, in a particle analyzer that fractionates particles appearing in a distribution map and analyzes the particles, the fractionation abnormality is easily determined, so that erroneous analysis is prevented and analysis accuracy is improved. Can be made to.

[Simple explanation of drawings]

[Figure 1]

It is a perspective view which shows the optical system which concerns on embodiment of this invention.

[Figure 2]

It is a system diagram which shows the fluid system which concerns on embodiment of this invention.

[Fig. 3]

It is a block diagram which shows the structure of the analysis part which concerns on embodiment of this invention.

[Fig. 4]

It is a display example of the distribution map which concerns on embodiment of this invention.

[Fig. 5]

It is a display example of the distribution map which concerns on embodiment of this invention.

[Fig. 6]

It is a display example of the distribution map which concerns on embodiment of this invention.

[Fig. 7]

It is a display example of the distribution map which concerns on embodiment of this invention.

[Fig. 8]

It is a system diagram which shows the detail of the main part of FIG.

[Fig. 9]

It is a flowchart which shows the operation of the blood analyzer of this invention.

[Explanation of symbols]

1 sheath flow cell 21 laser diode 22 Collimated lens 24 Condensing lens 25 pinhole board 26 photodiode 27 Condensing lens 28 Dichroic mirror 29 Photo Multiplier Tube 30 pinhole board 31 Photo Multiplier Tube 32 amp 33 amp 34 amp 35 Analysis Department 36 filters