JPH0251044A - Cell analyzer - Google Patents

Abstract

PURPOSE:To achieve a higher reliability of analysis with the removal of undesired information by a method wherein a histogram is prepared for one parameter detected by a cell optical information detecting means and a point of inflection is extracted therefrom to set a noise threshold. CONSTITUTION:A sample pump 7 is driven to send a sample to a feed liquid tube 8 while a feed liquid pump 9 sends a sheath liquid to a flow cell 10. A sheath flow is formed in a flow channel 10a to run in one row and cells are irradiated with a laser beam l one at a time to measure an intensity Io of forward scattered light, an intensity of 90 deg. scattered light and intensities of green and red fluorescence. The intensity 10 is inputted into a received light signal processing circuit 20a via a photo detector 15a and a circuit 20 extracts a point of inflection as given by noises due to a ghost and a dirt from a histogram to set a noise threshold Ns with a noise threshold circuit on the basis of the inflection point thus obtained. Thus, an undesired data is removed from the data to perform a recalculation thereby achieving a high efficiency with an automatic setting of an optimum noise threshold for the sample.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application The present invention relates to a cell analysis device using flow cytometry, and more specifically, to noise processing thereof.

(b) Conventional technology In flow cytometry, for example, a sample containing cells (or particles equivalent to this) labeled with a fluorescent dye is passed through a thin liquid stream together with a sheath liquid, and a hydrodynamic focusing effect is used to A beam of light such as a laser is irradiated onto each cell flowing in a row, and the intensity of scattered light and fluorescence generated by the cells is measured.
In other words, it instantaneously measures cell optical information and analyzes cells. Flow cytometry has the advantage of being able to analyze large amounts of cells at high speed and with high precision.

The cell analysis device that applies the above-mentioned flow cytometry includes a flow cell for forming a thin liquid flow, a light source (e.g., a laser) that irradiates a light beam to the cells flowing within the flow cell, and a light source (e.g., a laser) that irradiates the cells flowing within the flow cell. A photodetector that detects cellular optical information from cells and converts it into an electrical signal, a signal processing circuit that amplifies and integrates the signal of this photodetector, and a signal of cellular optical information that is output from this signal processing circuit. There are known devices that include a computer that performs arithmetic processing and the like.

For example, in the case of analysis of lymphocyte subsets in blood, blood that has been hemolyzed to remove red blood cells is further treated with a monoclonal antibody labeled with a fluorescent dye [in the case of two colors,
0KT4 monoclonal antibody labeled with fluorescein inthiocyanate (FITC: green fluorescence) and 0KT8 monoclonal antibody labeled with phyconilithrin (PE: red fluorescence)] are used as samples.

The sample cells flowing through the flow cell are irradiated with a laser beam, and four parameters are measured: the forward scattered light intensity;
. , 90° scattered light intensity■,. , green fluorescence intensity 19, and red fluorescence intensity 2 are detected by the detector, respectively, and cell optical information (hereinafter sometimes simply referred to as data) is obtained.

Since this sample contains monocytes, granulocytes, etc. in addition to lymphocytes, it is necessary to select data regarding lymphocytes from data regarding other cells.

Therefore, the forward scattered light intensity io and the 90° scattered light intensity ■ are considered to represent the size and internal structure of the cell. Create a site duram whose orthogonal coordinate axes are [see FIG. 5(a)].

In FIG. 5(a), b shows the distribution of lymphocytes, C shows the distribution of monocytes, and d shows the distribution of granulocytes. On this cytogram, an analysis area called a window is set, which includes the distribution of lymphocytes, for example, and lymphocyte data is sorted from the measured data (JP-A-62-1
(See Publication No. 34559). Regarding the data of the lymphocytes selected in this way, green fluorescence intensity ■9, red fluorescence intensity!
.

Arithmetic processing is performed to calculate the positive rate, etc.

(c) Problems to be Solved by the Invention In general, a sample contains minute dust and the like in addition to cells, and the sheath liquid may also contain minute dust. When these dust particles pass through the flow cell, unnecessary information, that is, noise is included in the cell optical information. For example, for analysis of lymphocyte subsets,
Red blood cell membrane components remaining as a result of hemolytic treatment appear in the sample as ghosts a, or noise e of dust in the sample or sheath fluid appears [see FIG. 5(a)].

Therefore, in conventional cell analyzers, a noise threshold circuit is provided in the signal processing circuit that processes the signal from the photodetector, and the noise threshold is set as shown in FIGS. 5(a) and 5(b). The reliability of the analysis is ensured by removing red blood cell ghosts (a) and noise (e) caused by dust. The noise threshold is set by an operator inputting the threshold level into the computer, and the computer transmitting the threshold level to the noise threshold circuit. In other words, the computer is only interposed between the operator and the noise threshold circuit;
In conventional cell analyzers, it can be said that noise is removed by hardware.

However, when a large number of samples are processed continuously, such as when using an automatic sample supply device (auto sampler), noise often occurs during measurement due to the effects of red blood cell ghosts and dust in the sheath fluid. There was a problem in that it was necessary to reset the threshold settings, which reduced inspection efficiency. Also, if you neglect to change the noise threshold,
If the change settings were inappropriate, there was a problem that the reliability of the analysis would decrease.

The present invention has been made in view of the above, and aims to provide a cell analysis device that can automatically set the most appropriate noise threshold for each sample.

(d) Means for Solving the Problems In order to solve the above problems, the cell analysis device of the present invention includes a flow cell through which a suspension of cells (or particles equivalent thereto) is flowed, and a cell flowing through the flow cell that is illuminated with light. a light source that irradiates a beam; a cell optical information detection means that detects cell optical information consisting of one or more parameters regarding cells irradiated with the light beam; a cell optical information selection means for selecting cell optical information of a target cell population from the cell optical information detected by the cell optical information detected by the cell optical information detection means and the cell optical information selection means; and an output means for outputting the processing results of the second cyto-optical information processing means, wherein the cyto-optical information is At least one of the cell optical information detected by the detection means
a histogram creation means for creating a histogram for two parameters; an inflection point extraction means for extracting an inflection point from the histogram created by the histogram creation means; , an unnecessary information removing means for setting a noise threshold and removing unnecessary information (noise) included in the cell optical information detected by the cell optical information detecting means.

(E) Function The function of the cell analyzer of the present invention will be explained using FIG. 3 and FIG. 5(b), taking analysis of a subset of lymphocytes as an example. FIG. 3 shows the forward scattered light intensity ■ created by the histogram creation means. The histogram is shown. The portion smaller than the inflection point p on this histogram corresponds to the noise caused by the ghost or dust.

Therefore, this inflection point p is extracted by the inflection point extracting means, and the unnecessary information removing means sets a noise threshold as shown in FIG. 5(b) based on the inflection point P. can be removed.

That is, the cell analyzer of the present invention removes noise using software, and can automatically set the most appropriate noise threshold for each sample.

(f) Example An embodiment of the present invention will be described below based on the drawings.

First, an overview of the cell analyzer according to the embodiment will be explained. This cell analyzer is suitable for analyzing blood samples, and the cell optical information is the forward scattered light intensity (■). , 90° scattered light intensity■,. , green fluorescence intensity I,, red fluorescence intensity! ,0
Four parameters are adopted. On the other hand, in the cell analyzer of this embodiment, noise thresholding is performed using hardware similar to the conventional method, and the software noise thresholding of the present invention is applied in such a manner that the noise thresholding is corrected. This software noise threshold is set based on the inflection point of the histogram of the forward scattered light intensity I0.

FIG. 1 is a diagram illustrating the configuration of the cell analysis device of this example. 2 is an autosampler, and its sample rack 2a is loaded with a plurality of sample containers 3, . . . 3. This sample rack 2a is driven by a drive mechanism (not shown) and positions a designated sample directly below the ship tube 4. Furthermore, this autosampler 2 is equipped with a shaking mechanism and a cooling mechanism (not shown), and shakes the sample at regular intervals.
The sample inside is kept at a low temperature (e.g. 4°C to 10°C).

Said ship tube 4 is connected to one boat of a three-way valve 5. Sample liquid feeding tubes 6a and 6b are connected to the other two boats of the three-way valve 5, respectively, and it is possible to connect the sample liquid feeding tube 6a and the ship tube 4, or the sample liquid feeding tubes 6a and 6b. can.

A sample pump 7 is provided at the other end of the sample liquid feeding tube 6a. On the other hand, the other end of the sample liquid feeding tube 6b opens into the sheath liquid feeding tube 8.

One end of the sheath liquid feeding tube 8 is connected to a liquid feeding pump 9, and the other end is connected to a flow cell 10. The flow cell 10 is made of quartz glass or the like, and a sheath flow is formed in the internal flow channel 10a, and due to the hydrodynamic focusing effect, cells or particles in the sample are aligned on the central axis of the flow channel 10a. be swept away.

The liquid flowing out from the flow cell 10 is guided to a waste liquid chape 11 and stored in a waste liquid tank 12.

Note that this cell analysis apparatus is equipped with a sheath liquid tank (not shown), and the sample pump 7 and liquid feed pump 9 are replenished with the sheath liquid. In addition, the liquid delivery system including the autosampler 2, pump 7.9, etc. can be sealed to prevent biohazards.

Around the flow cell 10, a laser (light source) 14, a photodetector (III cell optical information detection means) 15a, 15b1.1
5c and 15d are provided. The laser beam 2 from the laser 14 is irradiated onto the cells 1 or particles flowing through the flow channel 10a. Signal light is generated from the cell 1 or particle, and the signal light directed inward is focused on the lens 16a as forward scattered light and enters the photodetector 15a.

17 is a beam blocker that prevents the laser beam 2 from directly entering the photodetector 15a.

On the other hand, the signal light in the 90° direction from the cell 1 is transmitted through the lens 16.
The light is focused at b. A portion of this signal light is reflected by the dichroic mirror 18a and enters a photodetector 15b for detecting 90° scattered light. Dichroic mirror 18a
A portion of the signal light transmitted through the filter 1 is further reflected by another dichroic mirror 18b, and then passed through the filter 1.
The light passes through 9a and is received by a photodetector 15c for green fluorescence.

The signal light that has passed through the dichroic mirror 18b passes through a filter 19b and is received by a red fluorescence photodetector 15d. Note that, for example, the laser 14 may include an argon laser, a helium neon laser, and a photodetector 1 for forward scattered light.
5a has a photodiode, and other detectors 15b, 15
A photomultiplier tube is applied to c and 15d.

The light reception signals of the photodetectors 15a, 15b, 15c, and 15d are processed by light reception signal processing circuits 20a, 20b, and 20, respectively.
c, 20 d processed and analog/digital (
A/D) converted into digital data by converter 25 and stored in memory 2G.
is stored once. As shown in FIG. 2, the received light signal processing circuit 20a includes a preamplifier 21 that amplifies the received light signal of the photodetector 15a, an integrator 22 that integrates the output of the preamplifier 21, and an amplifier 23 that amplifies the output of the integrator 22. , and a noise threshold circuit 24 that does not pass the received light signal corresponding to noise among the outputs of the amplifier 23. The other light receiving signal processing circuits 20b, 20c, and 20d are also 20
It has the same configuration as a.

In this embodiment cell analyzer, signal processing circuits 20a, 2
The forward scattered light intensity ■ is determined by each noise threshold circuit 24 of 0b. , the noise threshold Nh shown in FIG. 5(a) is set on the cytogram of the 90° scattered light intensity I90. This noise threshold Nh is a hardware noise threshold, and is fixed in this embodiment.

The data stored in the memory 26 is taken in and processed by the MPU 27. The MPU27 calculates the forward scattered light intensity■
. A function to create a histogram of 10 histograms, a function to extract an inflection point P from these 10 histograms, a function to correct the noise threshold Ns using this inflection point p and remove noise,
It has a function of determining an analysis region for the data from which noise has been removed, a function of performing arithmetic processing on the data within this analysis region, a function of controlling the autosampler 2, the sample pump 7, the liquid pump 9, and the like.

A keyboard 28, a CRT 29, and a printer 30 are connected to the MPU 27. The keyboard 28 is used for selecting and setting protocols (measurement conditions) and other commands.
This is for inputting to the PU27. CRT29 is
It monitors measurement and calculation processing, and is equipped with a printer (
Output means) 30 is for printing out the data processing results of the MPU 27.

Next, the operation of the cell analyzer according to the embodiment will be explained. When the power of the cell analyzer is turned on, the MP
The program is read into U27 and the system starts up. Then, a protocol suitable for the sample to be measured is selected and set [Step (hereinafter referred to as ST) 1, see Figure 4]
This protocol includes the detection gain of the photodetector, the method of correction calculation, etc., but this is because the sample processing method differs depending on the measurement purpose. For example, the monoclonal antibody used for each processing Because the reaction with cells, etc. differs, it is necessary to change the detection gain of the photodetectors 15a, 15b, 15C115d, and the binding mode between each monoclonal antibody and fluorescent dye is different, so the correction calculation method also differs accordingly. It is necessary to change it accordingly.

When the STI processing is completed, the sample rack 2a is driven to position the sample container 3 containing the sample to be measured directly below the ship tube 4. Then, the ship tube 4 is lowered into the sample container 3, and the three-way valve 5 is switched so that the ship tube 4 and the sample liquid feeding tube 6a communicate with each other.
The sample pump 7 is driven to the suction side, and the sample is sucked into the sample liquid feeding tube 6a (Sr2).

Next, the three-way valve 5 is switched to communicate the sample liquid feeding tubes 6a and 6b, the sample pump 7 is driven to the liquid feeding side, and the sample is sent to the liquid feeding tube 8. On the other hand, the liquid feeding pump 9 is driven to the liquid feeding side, and the sheath liquid is sent to the flow cell 10. A sheath flow is formed within the flow channel IOa, and due to the hydrodynamic focusing effect, the cells flow in a line on the central axis of the flow channel 10a. Each of these cells is irradiated with laser beam 2, and the forward scattered light intensity is 1. ．． 90° scattered light intensity ■.

, green fluorescence intensity I9, and red fluorescence intensity ■<(Sr1).

When performing white blood cell analysis, forward scattered light intensity■. and 9
0° scattered light intensity I. A cytogram is created [see FIG. 5(a)]. Then, for example, an analysis area is set for the distribution of lymphocytes, the average intensity, standard deviation (SD), and coefficient of variation (CV) of the data within this analysis area are calculated, and these are compared with the input standard value. It is determined whether the data is abnormal or not (Sr1). When this determination is YES, the process branches to Sr1, and when this determination is NO (normal), the process branches to Sr1.

In Sr1, the operator is notified of the abnormality, and in Sr1,
Automatic correction of the noise threshold and analysis area is performed, and further recalculation is performed in Sr7.

In FIG. 5(a), a noise threshold Nh is set by the noise threshold circuit 24, but the noise e due to dust may not be removed by this noise threshold Nh alone. Therefore, the noise threshold is automatically corrected by setting the noise threshold Ns as shown in FIG. 5(b) through arithmetic processing within the MPU 27.

To set the noise threshold Ns, first, as shown in Fig. 3, the forward scattered light intensity ■. The inflection point p is extracted from the histogram (this histogram is for data from which noise has been removed by the noise threshold Nh). I from this inflection point p. The part where is small is noise due to ghosts and dust, so the fifth part is based on the inflection point P.
As shown in Figure (b), a noise threshold Ns is set, and unnecessary data is removed from the data. In Figure 3, the area larger than the inflection point p is the total number of white blood cells, so if the lymphocyte analysis area is set, the abundance ratio of lymphocytes in white blood cells can be determined by calculation, and the white blood cell This information can be useful when testing the immune function of patients. Note that since the present invention does not primarily involve automatic correction of the analysis area, a description thereof will be omitted (see, for example, the above-mentioned Japanese Patent Laid-Open No. 134559/1983).

If it is determined that there is no abnormality in Sr1, or if recalculation is performed in Sr7, the process advances to Sr1, where the data is processed in a predetermined format, displayed on the CRT 29, and sent to the printer 3.
Printed out from 0. At Sr9, it is determined whether or not there are any unmeasured samples among the specified samples. If this determination is YES, the process branches to 5TIO, the sample rack 2a is driven, and the next sample is transferred to the ship tube. 4 and returns to STI. On the other hand, the judgment in ST9 is N.
In the case of O, the measurement is ended.

In the above description, analysis of blood samples, particularly white blood cells, is taken as an example, but the present invention is also applicable to analysis of samples other than blood. Further, the parameters related to noise threshold setting are not limited to the forward scattered light intensity, and can be changed as appropriate.

(G) As described in detail of the invention, the cell analysis device of the present invention includes a histogram creation means for creating a histogram for at least one parameter of the cell optical information detected by the cell optical information detection means; An inflection point extraction means extracts an inflection point from the histogram created by the histogram creation means, and a noise threshold is set based on the inflection point extracted by the inflection point extraction means, and the cell optical information is detected. The present invention is characterized by being equipped with an unnecessary information removal means for removing unnecessary information contained in the cell optical information detected by the method, so that the optimal noise threshold for the sample can be automatically set, and the analysis can be performed easily. This has the advantage of increasing efficiency and reliability of analysis.

[Brief explanation of the drawing]

FIG. 1 is a diagram illustrating the configuration of a cell analyzer according to an embodiment of the present invention, FIG. 2 is a block diagram illustrating the configuration of a signal processing circuit of the cell analyzer, and FIG. A diagram showing a histogram of the forward scattered light intensity in the cell analyzer, FIG. 4 is a flow diagram explaining the operation of the cell analyzer,
FIGS. 5(a) and 5(a) are cytograms of forward scattered light intensity and 90′ scattered light intensity, respectively, for explaining the noise threshold. 10: Flow cell, 14: Laser, 15a15b
15cm15d: Photodetector, 27: MPU,
29: CRT. 30: Printer. Figure Figure Figure Procedure amendment (voluntary) Name of the invention Cell analyzer Address 4, Agent address 10 Hanazono Tsuchido-cho, Ukyo-ku, Kyoto Representative Yoshio Tateishi 604 3-3 Mibu Kayo Gosho-cho, Nakagyo-ku, Kyoto 1 Kyoto Muro Building 5F Voluntary Amendment 7, Contents of Amendment (1) The "Claims" of the specification will be amended as shown in the attached sheet. (2) From the 4th line to the 5th line on page 3, the 13th
"Cell optical information" in the lines 1 and 16 is corrected to "cell optical information j. (3) From line 20 on page 3 to line 6 on page 4 - It says, "Hemolytic treatment...is used."
“A monoclonal antibody labeled with a fluorescent dye (in the case of two colors, fluorescein inthiocyanate) (FITC)
: green fluorescence) labeled monoclonal antibody and phyconilithrin (PE: red fluorescence) labeled monoclonal antibody]
After reacting with blood, this is hemolyzed and used as a sample. ” he corrected. (4) In the 11th line of page 4, the phrase "cell optical information" is corrected to "cell optical information." (5) In the 12th line of page 5, the phrase "Cytoscopic optical information" is corrected to "Cytoscopic optical information." (6) From line 16 to line 17 on page 5, “
The phrase ``noise e'' is corrected to ``noise e, etc.''. (7) From page 7, line 11 to page 8, line 14, the phrase ``This light beam is...'' is replaced by A cell light information detection means for detecting cell light information consisting of one or more parameters regarding cells; and from the cell light information detected by the cell light information detection means,
A cell light information sorting means for sorting out cell light information of a target cell population; and a cell for processing the cell light information detected by the cell light information detection means and the cell light information selected by the cell light information selection means. A histogram of at least one parameter of the cell light information detected by the cell light information detection means is provided, comprising a light information processing means and an output means for outputting a processing result of the cell light information processing means. A histogram creation means to create, an inflection point extraction means to extract an inflection point from the histogram created by this histogram creation means, and a noise threshold set based on the inflection point extracted by this inflection point extraction means. The present invention is characterized by further comprising an unnecessary information removing means for removing unnecessary information (noise) contained in the cell light information detected by the cell light information detecting means. ” he corrected. (8) From the first line to the second line of page 9, the phrase ``due to dust'' is corrected to ``due to dust, etc.''. (9) In the 16th line of page 9, the phrase "Cytoscopic optical information" is corrected to "Cytoscopic optical information." (10) Replace rST4J on page 16, line 16 with rsT3. and correct it. (11) rST5J on the 4th line of page 17,
Correct with rsT4J. (12) In the 13th line of page 17, the phrase ``noise e'' is corrected to ``noise e, etc.''. (13) Replace "ST5" on page 18, line 15 with rST4. and correct it. (I4) Page 19, line 13, lines 13 to 14
In the 19th and 20th lines, "Cytoscopic optical information J" is corrected to "Cytoscopic optical information J." 8. Transcript of attached documents (1) Scope of claims One or more documents stating <Patent Application No. 63-202410> 2. Scope of Claims: (1) A flow cell through which a cell suspension is flowed, and a light source that irradiates a light beam to the cells flowing within the flow cell; For the cells irradiated with this light beam, 1 or 2
From the above-mentioned parameter detection means for detecting Plate Steal
A target cell selection means for selecting the target cell population, and a target and criminal selection means for selecting the noble thief and the thief detected by the target and detection means. In a cell analyzer comprising a processing means for processing a calculation result of the stolen information, and an output means for outputting the processing result of the processing means, the information on the accused person is A histogram creation means for creating a histogram for one parameter of the number of tribute people detected by the detection means, and an inflection point extraction for extracting an inflection point from the histogram created by the histogram creation means. and a noise threshold is set based on the inflection point extracted by the inflection point extraction means, and the U detected by the U detection means is set.
A cell analysis device comprising: unnecessary information removal means for removing unnecessary information included in the cell analyzer. Patent applicant Tateishi Electric Co., Ltd. Agent Patent attorney Shigeru Nakamura

Claims (1)

[Claims] (1) A flow cell through which a cell suspension is passed, a light source that irradiates a light beam onto the cells flowing within this flow cell, and a cell optical information consisting of one or more parameters regarding the cells irradiated with this light beam. A cell optical information detection means for detecting cell optical information; a cell optical information sorting means for selecting cell optical information of a target cell population from the cell optical information detected by the cell optical information detection means; a cell-optical information processing means for computationally processing the cell-optical information detected by the cell-optical information detection means and the cell-optical information selected by the cell-optical information selection means; and processing by the cell-optical information processing means. an output means for outputting a result; a histogram creation means for creating a histogram for at least one parameter of the cell optical information detected by the cell optical information detection means; and the histogram creation means. an inflection point extraction means for extracting an inflection point from the histogram created by the inflection point extraction means; and a noise threshold is set based on the inflection point extracted by the inflection point extraction means, and the noise threshold is detected by the cell optical information detection means. A cell analysis device comprising unnecessary information removal means for removing unnecessary information contained in the cell optical information obtained.