JP2002188993A - Particle analyzer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To calculate a differential integral value of a photodetection signal waveform as a parameter for discriminating a particle of simple shape and structure from a particle of complicated shape and structure or close passing of the particle to enhance classification precision. SOLUTION: The differential integral value of the signal waveform is found by sampling and A/D-converting the photodetection signal waveform of the particle, and by adding cumulatively absolute values of differences between neigboring sampling data. A scatter program, a histogram or the like is prepared using the differential integral value of the signal waveform, and other parameter such as a peak level and a pulse width, to classify the particles.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a particle analyzer for analyzing particles such as blood cells and various cells contained in blood and urine.

[0002]

2. Description of the Related Art Various particle analyzers have been used to analyze particles such as cells and bacteria contained in blood cells and urine in blood. A flow cytometer is known as such a particle analyzer. In a general flow cytometer, a sample liquid containing particles to be analyzed is wrapped in a sheath liquid, narrowed down narrowly in a sheath flow cell, the particles are aligned and passed, and a laser beam is irradiated there, and each particle is irradiated. Obtain optical information such as scattered light and fluorescence. The obtained optical information is converted into a light detection signal by a photoelectric conversion element, and a parameter representing a characteristic of the particle is calculated by processing the signal waveform. Based on the parameter, the particles to be analyzed are classified and counted.

From the light detection signal, parameters such as a signal waveform height (peak level) and a signal waveform width (pulse width) are calculated. For example, the peak level of the forward scattered light detection signal indicates the size of the particle. The pulse width of the forward scattered light detection signal indicates the particle length. Meanwhile, particles in advance,
For example, when a nucleated cell is subjected to fluorescent staining, the peak level of the fluorescence detection signal indicates the degree of staining of the nucleus and the like, and the pulse width indicates the length of the fluorescently stained portion. By creating a histogram based on such parameters representing the characteristics of each particle, or by creating a scattergram showing the distribution of particles by combining a plurality of parameters, the type and number of particles contained in the sample Statistical analysis has been performed.

[0004]

By the way, a light detection signal waveform detected from a particle having a simple form and structure in a flow cytometer has a simple waveform as shown in FIG.
On the other hand, a particle having a complicated shape and structure also has a complicated signal waveform, and has one or more valleys. Further, even in the case of particles having a simple form and structure, when a plurality of particles pass through the detection area of the flow cytometer in a state of being close to each other (close passage), similar to the particles having a complicated form and structure, A valley occurs in the signal waveform. FIG. 8 shows an example of such a signal waveform.

However, parameters such as the peak level and pulse width do not reflect the state of the valley of the signal waveform. Sometimes they cannot be distinguished. When a plurality of particles pass close to each other, they may not be distinguished from large particles having complicated shapes and structures.

In view of these problems, the present invention provides a particle analyzer having a function of calculating a parameter reflecting a valley of a signal waveform in addition to a conventional peak level and pulse width.

[0007]

According to the present invention, there is provided a detecting unit for flowing a sample liquid containing particles into a sheath flow cell, irradiating the sheath flow cell with laser light to detect a light detection signal from the particles, and a detecting unit. A signal processing unit that processes the signal waveform of the photodetection signal detected in the calculation of parameters representing the characteristics of the particles, and an analysis unit that analyzes the particles based on the parameters calculated by the signal processing unit. In the particle analyzer provided with the above, there is provided a particle analyzer in which the signal processing unit calculates a difference integral value of the signal waveform as a parameter.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Particles to be analyzed in the present invention include particles such as blood cells in blood and cells and bacteria in urine.

The detecting section wraps a sample liquid containing particles in a sheath liquid, flows the same through a sheath flow cell, irradiates the sheath flow cell with laser light, and detects scattered light, fluorescence, and the like from each particle as a light detection signal. belongs to. A known optical detector can be used for the detection unit. Specifically, the sheath flow cell is preferably transparent and has a smooth surface in order to obtain optical information from particles passing through the inside thereof, and a material such as glass is used. A semiconductor laser, an argon laser, or the like is used as the laser light for irradiation. A photodiode, a phototransistor, a photomultiplier tube, or the like is used as a photoelectric conversion element for photoelectrically converting optical information from particles to detect a light detection signal. In addition, various optical components such as lenses and mirrors are used as needed.

The signal processing section comprises a circuit for processing the waveform of the light detection signal for each particle obtained by the detection section and calculating various parameters including a difference integral value. In addition to the difference integrated value, a peak level, a pulse width, or the like may be calculated as a parameter. Peak level,
Parameters such as the pulse width can be calculated by a known peak hold circuit, counter circuit, or the like.

In the present invention, the difference integral value is a value obtained by differentiating a signal waveform and adding its absolute value. That is, the difference integrated value of a signal having no valley in the waveform as shown in FIG. 7 is equal to a value obtained by doubling the peak level H of the signal.

On the other hand, the difference integrated value of a signal having a valley in the waveform is larger than a value obtained by doubling the peak level of the signal. An example of the calculation will be described with reference to FIG. That is, FIG.
Is obtained by the sum of H1, H2, H3, and H4, the following equation holds. (H1 + H2 + H3 + H4) = (H1 × 2 + H3 × 2)
> (H1 × 2)

Since H1 is the peak level of the signal shown in FIG. 8, it can be seen from the above equation that the difference integrated value of the signal having a trough in the waveform is larger than the value obtained by doubling the peak level of the signal. . The difference integrated value becomes larger as the waveform has more valleys and deeper, and the difference from the value obtained by doubling the peak level becomes larger.

The nature of the difference integral value as described above indicates that the difference integral value of the signal waveform is useful as a parameter that reflects the complexity of the morphology and structure of the particle or the near passage of the particle.

The difference integral value of the signal waveform can be calculated by sampling the signal at a constant cycle and using a circuit for cumulatively adding the absolute value of the difference between adjacent data. In the conventional analog signal processing method, the circuit becomes complicated, and the temperature change and the secular change of the signal gain are apt to occur, so that it is difficult to secure the reliability of the difference integrated value. Therefore, it is preferable that the analog signal is sampled and A / D converted at a frequency sufficiently higher than the signal frequency band, and that the waveform data is digitally processed to calculate the difference integral value.

If the S / N ratio of the detected signal is poor, a high-frequency noise signal is mixed, so that even a signal of a particle having a simple form and structure contains many minute valleys in the waveform. This makes it difficult to distinguish particles having complicated shapes and structures. Therefore, in order to effectively use the difference integral value as a parameter, it is necessary to remove a noise signal from the detected signal. As a countermeasure, a method of providing a high-cut filter as a pre-process before the process of calculating the difference integral value in the signal processing unit can be considered.

The analyzing section analyzes particles present in the sample liquid by performing statistical analysis based on various parameters calculated by the signal processing section, and can be constituted by a microcomputer, a personal computer, or the like. At the time of statistical analysis, a histogram, a scattergram formed by combining a plurality of parameters, or the like may be created.

The present invention may further include an output unit for outputting an analysis result by the analysis unit. A display unit such as a CRT or an LCD, a printer, or the like can be used for the output unit.

[0019]

Embodiments of the present invention will be described below with reference to the drawings. The present invention is not limited to the following embodiments.

FIG. 1 is a diagram showing the configuration of this embodiment. In the detection unit 1, the laser light emitted from the laser light source 11 is squeezed into an elliptical shape by the condenser lens 12 and is applied to particles passing through the sheath flow cell 13. In the case where urine is used for the sample liquid, the size of the ellipse is approximately the same as the diameter of the particle to be detected in the direction of flow of the sample liquid, for example,
Before and after, in the direction orthogonal to the flow direction of the sample, the diameter is sufficiently larger than the diameter of the particle to be detected, for example, about 150 to 400 μm.

Thereafter, the forward scattered light is detected by the photodiode 15 via the condenser lens 14 and sent to the signal processing section 2 as a forward scattered light signal. The side scattered light reflected by the dichroic mirror 17 from the sheath flow cell 13 via the condenser lens 16 is supplied to the photomultiplier tube 18, and the side fluorescence passed through the dichroic mirror 17 is supplied to the side by the photomultiplier tube 19. The scattered light signal and the side fluorescence signal are detected and sent to the signal processing unit 2.

Hereinafter, a circuit for calculating the difference integral value in the waveform processing in the signal processing unit 2 will be described. The signal processing unit 2 of the present embodiment also has a circuit for calculating parameters such as a peak level and a pulse width, but these are well known and will not be described.

FIG. 2 is a diagram showing a difference integral value calculation circuit of the signal processing unit 2. FIG. 3 is a schematic timing chart of the present circuit. In the difference integral value calculation circuit according to the present embodiment, the absolute value of the difference between adjacent data of the signal waveform sampling data of the portion (particle signal) where the particle is detected in the continuous light detection signal waveform is cumulatively added. Find the difference integral value.

First, the A / D converter 21 performs sampling A / D conversion on the light detection signal (analog signal) detected by the detection unit 1. The sampling clock used at this time has a frequency sufficiently higher than the frequency band of the analog signal. A for each sampling cycle
The / D-converted waveform sampling data D1 is compared with a preset discrimination level in the comparator 22 in order to detect whether or not the signal is a particle signal. It is assumed that particles have been detected, and the comparator output signal S1 becomes HIGH. At the same time, the waveform sampling data D1 is latched in the sampling data register 23 in synchronization with the sampling clock.

To the differentiator 24, the waveform sampling data D1 from the A / D converter 21 and the waveform sampling data D2 latched in the sampling data register 23 are sent. Here, since the data D2 latched in the sampling data register 23 is one clock before the data D1 from the A / D converter 21, the absolute value of the difference between these two waveform sampling data is calculated by the differentiator 24. Is calculated.

When the comparator output signal S1 becomes HIGH, the control circuit 25 operates to generate an addition enable signal S2. As a result, the accumulator 26 for adding the differentiator output data D3 successively output from the differentiator 24 is added.
Operation starts. When the waveform sampling data D1 becomes smaller than the discrete level and the comparator output signal S1 of the comparator 22 becomes LOW, the control circuit 25 responds accordingly.
Becomes low, and the operation of the accumulator 26 stops.

The output data D4 of the accumulator 26 is a difference integrated value during a period when the waveform sampling data D1 is larger than the discrete level. Then, by the adder 27,
Further, a double value of the discrete level is added. The data (adder output data D5) is synchronized with the latch enable signal S3 output from the control circuit 25 in response to the addition enable signal S2 being LOW, and is latched by the difference integration register 28.

When the latching of the difference integrated value of one particle signal into the difference integration register 28 is completed, the accumulator 26 is cleared to prepare for the next particle signal.
This clear operation is based on the clear signal S4 output from the control circuit 25 in response to the latch enable signal S3.

The difference integrated values of the respective particle signal waveforms calculated in the above steps are stored in a memory (not shown) in time series together with separately calculated parameters such as a peak level and a pulse width.

When the measurement for one sample is completed, the difference integral value and other parameters stored in the memory are read out, and the analysis unit 3 creates a scattergram,
Statistical analysis is performed.

FIG. 4 is an example of a scattergram created when urine is used as a sample solution containing particles to be analyzed in the particle analyzer using this embodiment, and the vertical axis of the coordinate indicates forward scatter. 1/2 of the difference integrated value of the light detection signal waveform
The horizontal axis indicates the peak level of the waveform of the forward scattered light detection signal.

The difference integrated value of a signal having no valley in the waveform is the same as a value obtained by doubling the peak level of the signal, and the difference integrated value of a signal having a valley in the waveform has a peak level of 2
As described above, the value becomes larger than the multiplied value. On the scattergram of FIG. 4 utilizing these facts, simple particles having a morphology / structure are distributed near a straight line having a slope of 1, and particles distributed above this straight line are morphology / structure.
It can be determined that particles having a complicated structure or a plurality of particles have passed close to each other.

Examples of particles contained in urine include various cells such as erythrocytes, leukocytes, epithelial cells, casts, streptococci, and bacteria. Among them, erythrocytes, leukocytes, bacteria, etc. are particles having a simple form and structure and are likely to be distributed in the region 4a, and particles having a complicated form and structure such as cylinders and streptococci, or simple forms and structures. When a large particle passes close to the detection unit, it is highly possible that the particle is distributed in the region 4b.

As described above, by using the difference integral value of the signal waveform as a parameter, it is possible to classify a particle having a simple form and structure from a complicated particle, or a particle that has passed closely. In addition, by using the results of this analysis in combination with the results of conventional analysis,
Particle classification accuracy can be improved.

By using only the peak level and the difference integral value as parameters, it may be difficult to distinguish between a particle having a complicated form and structure and a case where a simple particle has passed close. FIG. 5 shows a comparison between the waveform of the forward scattered light detection signal of the cylinder and the waveform of the forward scattered light detection signal when two leukocytes pass close to each other. These are shown in the scattergram of FIG. All appear in the area 4b.

However, in the particles contained in urine,
Particles having complicated morphology and structure have a large size and a very large pulse width, and are generally larger than the pulse width when a simple particle having a simple morphology and structure passes closely. For example, the particle length of a cylinder is usually 10
About 0 to 300 μm, and the particle length of leukocytes is 10 to 1
Since it is about 5 μm, the pulse width of the cylinder and the pulse width when white blood cells pass close to each other are clearly different from each other as shown in FIG. Therefore, by adding analysis using the pulse width as a parameter to the analysis using the difference integrated value and peak level, the classification accuracy between particles with complicated morphology and structure and those when simple particles pass close to each other is improved. can do.

First, in the analysis unit 3, an area 4b is assumed on the scattergram of FIG. 4 in which particles having complicated morphology and structure and particles having simple morphology and structure are mixed. . Next, a histogram using the pulse width as a parameter or a scattergram using the pulse width and the peak level as parameters is created for the particle group distributed in the region.

FIG. 6 shows an example of the histogram thus created. The vertical axis of the coordinates indicates the frequency of the particles, and the horizontal axis indicates the pulse width. The area 4b of the scattergram in FIG.
Is calculated for each pulse width. Here, by setting a threshold value corresponding to the type of particle in advance, it is possible to count and classify particles each having a complicated form and structure and particles that have passed close to each other. In addition, by using this result in combination with the results of conventional analysis,
Particle classification accuracy can be improved.

[0039]

As described above, according to the present invention, the difference integral value is used as a parameter reflecting the characteristics of particles,
Particles can be classified from the viewpoint of the complexity of the morphology and structure of the particles or the close passage of a plurality of particles.

Further, the particle analyzer of the present invention enables particle analysis with higher classification accuracy by using the difference integral value in combination with conventional parameters such as a peak level and a pulse width.

[0041]

[Brief description of the drawings]

FIG. 1 is a diagram showing a schematic configuration of the present invention.

FIG. 2 is a block diagram showing a difference integral value calculation circuit;

FIG. 3 is a diagram showing a timing chart in a differential integration value calculation circuit.

FIG. 4 is a diagram showing a scattergram created in the present invention.

FIG. 5 is a diagram comparing a signal waveform of a cylinder with a signal waveform when white blood cells pass close to each other.

FIG. 6 is a diagram showing a histogram according to the embodiment.

FIG. 7 is a diagram illustrating an example of a signal having no trough in a waveform;

FIG. 8 is a diagram showing an example of a signal having a trough in a waveform.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Detection part 11 Laser light source 12 Condenser lens 13 Sheath flow cell 14 Condensing lens 15 Photodiode 16 Condensing lens 17 Dichroic mirror 18 Photomultiplier tube 19 Photomultiplier tube 2 Signal processing part 21A / D converter 22 Comparator 23 Sampling data Register 24 Difference device 25 Control circuit 26 Cumulative adder 27 Adder 28 Difference integration register D1 waveform sampling data D2 waveform sampling data D3 difference device output data D4 Cumulative adder output data D5 adder output data S1 comparator output signal S2 addition enable Signal S3 latch enable signal S4 clear signal 2 signal processing unit 3 analysis unit

Claims (4)

[Claims] 1. A detection unit for flowing a sample liquid containing particles through a sheath flow cell, irradiating the sheath flow cell with laser light to detect a light detection signal from the particles, and detecting a light detection signal detected by the detection unit. A signal processing unit that processes a signal waveform to calculate a parameter representing the characteristics of the particles, and an analysis unit that analyzes the particles based on the parameters calculated by the signal processing unit. The particle analyzer according to claim 1, wherein the signal processing unit calculates a difference integral value of the signal waveform as a parameter. 2. The signal processing section performs sampling A / D conversion on a photodetection signal detected by the detection section at a cycle higher in frequency than its frequency band, and cumulatively adds the absolute value of the difference between adjacent data. The particle analyzer according to claim 1, wherein a difference integral value of the signal waveform is calculated by performing the calculation. 3. The particle analyzer according to claim 1, wherein the signal processing unit includes a high-cut filter as a pre-process of a process for calculating a difference integral value. 4. The signal processing unit further calculates a peak level of a signal waveform as a parameter, and the analysis unit creates a scattergram based on a difference integrated value of the signal waveform and a peak level of the signal waveform. The particle analyzer according to claim 1, wherein