JPH0242358A - Cell analyzer - Google Patents

Abstract

PURPOSE:To determine the fraction meeting the distribution of a desired cell group in a short processing time by extracting the max. frequency value point on the cytogram of parameters relating to the fraction of the cell groups. CONSTITUTION:A cell light information collecting means collects the cell light information of the desired cell group for the fraction B obtd. by a cell group fractionation means; thereafter, the max. frequency value point (q) is extracted on the cytogram and straight lines l1-l12 are formed radially from this point (q). The histogram on the straight lines l1-l12 is as shown in, for example, the figures (a), (b). The points where the histogram intersects with the prescribed threshold values or the inflection points X, etc., of the histogram are extracted as boundary lines (r). The fraction B' which fits better to the distribution of the desired cell group is obtd. if the boundary points r1-r12 of these light information on the unnecessary cells, etc., into the collected cell light information is obviated if the light information on the desired cell group is further collected in accordance with this fraction. The analysis accuracy is thus improved.

Description

[Detailed Description of the Invention] (a) Industrial Application Field The present invention relates to a cell analysis device that applies flow cytometry, and more specifically, it selects and collects cell optical information about a specific cell population in a sample. Regarding processing.

(b) Conventional technology Flow cytometry involves, for example, passing cells (or similar particles) labeled with a fluorescent dye into a thin liquid stream.
Using a hydrodynamic focusing effect, a laser beam is irradiated onto each cell flowing in a line, and the intensity of scattered light and fluorescence generated by the cells, in other words, cell optical information, is instantly measured and analyzed. It is something to do. 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;
There are known devices equipped with a computer that performs analysis and processing of the cell optical information converted into electrical signals.

This conventional cell analysis device flows a cell suspension (sample) stained with a fluorescent dye into a flow cell together with a sheath liquid. A sheath flow is formed within the flow cell, and cells flow in a line along the central axis of the flow cell due to hydrodynamic focusing effects.

The intensity of scattered light and fluorescence generated when these cells are irradiated with a light beam is detected by a photodetector such as a photomultiplier tube as parameters constituting cell optical information.

Now, when a plurality of cell populations are contained in a cell suspension, there are cases where it is desired to perform an analysis on one of these cell populations. For example, in the case of analysis of lymphocyte subsets in human blood, hemolyzed blood is used as a sample, but this sample contains monocytes and granulocytes in addition to lymphocytes. Therefore, it is necessary to select lymphocytes from the cyto-optical information obtained by measurement.

Therefore, a so-called window method is known as a method for selecting and collecting cell optical information of a necessary cell population (see, for example, Japanese Patent Laid-Open No. 134559/1983).

This window method uses one or two of the cell optical information.
An operator sets an analysis region called a window in a space using the above parameters as a coordinate system, and optical information of cells belonging to this region is collected as cell optical information of a target cell population.

For example, in the case of analysis of the lymphocyte subsets mentioned above,
Among cell optical information, 90° scattered light intensity ■. and forward scattered light intensity■. The following two parameters are used. Figure 6 shows ■. On the horizontal axis, ■. A schematic diagram of the site Durham is shown with the vertical axis taken as the vertical axis. b shows the distribution of lymphocytes, C shows the distribution of monocytes, and d shows the distribution of granulocytes. Note that a indicates the distribution of debris, which is composed of membrane components of red blood cells and the like, and is usually removed at the noise processing stage.

To analyze lymphocytes, a control sample (sample from a healthy individual) is used and a window e is set as shown by the two-dot chain line in FIG. Collecting the cell optical information belonging to this window e from the measured whole cell optical information,
Analysis processing is performed on fluorescence characteristics, and the results are displayed on a CRT and output from a printer.

However, with the window method, depending on the sample, it may not be possible to collect cell optical information of a target cell population unless the operator changes the window. For example, in the analysis of lymphocyte subsets, the position, size, and shape of the lymphocyte distribution shown in FIG. 6 change depending on the sample, and the operator must change the window e accordingly. Such window changes prevent many samples from being measured automatically and efficiently.

Therefore, the applicant has already filed an application for a cell analysis device that can automatically collect cell optical information without the need for the operator to set a window (Japanese Patent Application No. 62-2
No. 2884). This cell analysis device creates histograms for one or more parameters of cell optical information, extracts inflection points of these histograms, fractionates cell populations based on these inflection points, and Or, using two or more fractions as analysis regions, optical information of cells belonging to these fractions can be analyzed.
Collect as cyto-optical information of the target cell population.

For example, in the case of analysis of the lymphocyte subsets described above, 9
0° scattered light intensity I,. and forward scattered light intensity■. Create a histogram of [see Figure 7 (a) and Figure 7 et al.]
. And... From the histogram, the inflection point P+, p
g% P3, I. According to the histogram, the inflection point is 94%.
Extract P%. These inflection points p1 to p5 are I. , ■
. When expressed on a cytogram, it is fractionated into the fractions indicated by broken lines in FIG. Since the distribution of lymphocytes is contained in fraction B, the optical information of cells belonging to this fraction B, ie, ■.

is p8 or more and Pz or less, and ■. Optical information of cells in which p4 or less is p4 or less is searched from the cytooptical information of all the measured cells, and the cytooptical information of lymphocytes is collected.

(C) Problems to be Solved by the Invention In the conventional cell analysis device described above, the cytooptical information collection means collects the cytooptical information of the target cell population based on the fraction, but this fraction As shown in the figure, it is a rectangular area on the cytogram. Therefore, this fraction does not perfectly match the shape of the distribution of the target cell population, and unnecessary optical information of cells is included in the collected optical information of the target cell population, reducing analysis accuracy. There was a problem with the decline.

A contour tracing method is known as a method for finding an analysis region that more closely matches the shape of the distribution of the target cell population, but it requires time for calculation processing and is not preferred from the standpoint of efficiency of analysis.

The present invention has been made in view of the above, and aims to provide a cell analysis device that can determine a fraction that more closely matches the distribution of a target cell population in a short processing time.

(d) Means for Solving the Problems In order to solve the above problems, the cell analyzer of the present invention includes a flow cell through which a suspension of cells (including particles equivalent thereto) is flowed, and a cell flowing through the flow cell. a light source that irradiates a light beam; a cell optical information detection means that detects cell optical information consisting of a plurality of parameters for each cell irradiated with the light beam; and a cell optical information detection means that detects cell optical information consisting of a plurality of parameters. , cell population fractionation means for fractionating the cell population in the cell suspension based on one or more parameters;
Of the fractions obtained by this cell population fractionation means, 1 or 2
a cyto-optical information collecting means for collecting cyto-optical information of a target cell population based on the above fractions; and the cyto-optical information detected by the cyto-optical information-detecting means and the cyto-optical information. A cell optical information processing means for processing the cell optical information of the target cell population collected by the collection means, and an output means for outputting the processing result of the cell optical information processing means, the cell a maximum numerical value point extracting means for extracting the highest numerical value point on a cytogram of a parameter related to the fractionation of the cell population with respect to the cell optical information of the target cell population collected by the optical information collecting means; gram, a straight line forming means forms a straight line radially from the highest numerical value point extracted by the highest numerical value point extracting means, and a histogram on each straight line formed by this straight line forming means, the boundary is determined. A boundary point extraction means for extracting points and a region formed by connecting the boundary points extracted by this boundary point extraction means are defined as a final fraction, and based on this final fraction, cytooptical information of the target cell population is obtained. The present invention is characterized by comprising a second cell optical information collecting means for collecting.

(E) Function The function of the cell analyzer of the present invention will be explained using FIGS. 1 and 2 corresponding to the embodiment. After the collection means collects the cell optical information of the target cell population, the highest numerical value point q is extracted on the cytodaram (see Figure 1), and a straight line ff1l""/! is radiated from this point 9. I2 is formed. This straight line! 1-21. The above histograms are as shown in FIGS. 2(a), (b), and (C), for example. By extracting the point where this histogram intersects with a predetermined threshold value or the inflection point Fraction B with a better fit of the distribution.

is obtained. If optical information of the target cell population is further collected based on this fraction, optical information of unnecessary cells, etc. will not be included in the collected cell optical information, and an improvement in analysis accuracy can be expected.

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

This embodiment of the cell analyzer uses forward scattered light intensity ■ as cell light information (hereinafter simply referred to as data). ,9
0° scattered light intensity I,. , fluorescence intensity of two different colors ■7, r
, a total of four parameters are adopted.

FIG. 2 is a diagram showing the configuration of an example cell analysis device.

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 sample suction 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 and keeps the sample in the loaded sample containers 3, . . . , at a low temperature (for example, 4°C to 10°C).

The sample suction 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 ports of the three-way valve 5, respectively.
The sample liquid feeding tube 6a and the sample suction tube 4, or the sample liquid feeding tubes 6a and 6b can be communicated with each other. 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 tube 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 (cell optical information detection means) 15a, 15b1.15c
, 15d are arranged. A laser beam 1 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 l 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 guichroic mirror 18a and enters a photodetector 15b for detecting 90° scattered light. Guicroic mirror 18a
A part of the signal light that has passed through the filter 1 is further reflected by another gichroic 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 light from the guichroic mirror 18b passes through the filter 19b and is received by the red fluorescence photodetector 15d. In addition,
For example, an argon laser or a helium neon laser is used as the laser 14, a photodiode is used as the photodetector 15a for forward scattered light, and a photomultiplier tube is used as the other detectors 15b, 15c, and 15d.

The light reception signals of the photodetectors 15a, 15b, 15c, and 15d are converted into digital signals by an analog/digital converter (not shown), and then taken into the MPU 21. MPU2
1 is the forward scattered light intensity! . , 90° scattered light intensity I,. A function to create a cytogram and histogram for each, a function to extract inflection points from this histogram and divide the cell optical information into fractions, a function to collect the cell optical information belonging to this fraction, Additionally, the ability to determine the final fraction for this collected cellular optical information;
It has functions such as collecting cell optical information belonging to this final fraction and analyzing fluorescence characteristics, and controlling the sample pump 7, liquid feeding pump 9, and autosampler 2.

A keyboard 22, a CRT 23, and a printer 24 are connected to the MPU 21. The keyboard 22 is used for selecting and setting protocols (measurement conditions) and other commands.
This is for inputting to the PU21. CRT23 is
It monitors the measurement, and the printer (output means) 2
Reference numeral 4 is for printing out the processing results of the MPU 21, such as a cytogram or a histogram.

Next, the operation of the cell analyzer according to the embodiment will be explained.

First, samples that have been processed according to the purpose of analysis are placed in sample containers 3 and loaded onto the sample rack 2a. For example, samples for lymphocyte subset analysis are
0KT4 monoclonal antibody labeled with fluorescein isothiocyanate (FITC) and 0KT labeled with phyconilithrin (PE) were added to hemolyzed patient blood.
8 monoclonal antibody.

When the cell analyzer is powered on, the ROM (not shown)
The program is read into the MPU 21 and the system starts up [Step (hereinafter referred to as ST) 1, Fig. 4 (a)
reference〕. Next, a protocol suitable for the sample to be measured is selected and set. In this protocol, the photodetector 15a
, 15b, 15c, and 15d, the contents include measurement conditions such as detection gains and correction calculations. This is because the sample processing methods differ depending on the purpose of measurement, and for example, the reactivity of monoclonal antibodies used for each treatment with cells is different.

It is necessary to change the detection gains of 15c and 15d, and since the binding mode between each monoclonal antibody and the fluorescent dye is different, the correction calculation also needs to be changed accordingly.

When the Sr2 treatment is completed, the sample rack 2a is driven, and the sample container 3 containing the sample to be measured first is positioned directly below the sample suction tube 4. And three-way valve 5
is switched so that the sample suction tube 4 and the sample liquid supply tube 6a are in communication with each other, the sample suction tube 4 is lowered into the sample container 3, the sample pump 7 is driven to the suction side, and the sample is transferred to the sample liquid supply tube 6a. 6a (Sr3).

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 10a and flows in a line on the central axis of the cell flow channel loa due to the hydrodynamic focusing effect. Each of these cells is irradiated with a laser beam l, and the forward scattered light intensity is 1. ．． 90° scattered light intensity ■. , green fluorescence intensity I9, red fluorescence intensity! , are measured respectively < (Sr1).

In Sr5, the MPU 21 has forward scattered light intensities of 1, . 90
``Cytogram with scattered light intensity [1,.'' as the orthogonal axis (6th
(see figure), and their respective histograms [Figure 7 (a)
(b)] is created. And in Sr6, ■,
. It is determined whether an inflection point can be extracted from the histogram.

■, if the sample is normal. From histogram 3
Three inflection points P+, 9z, and P3 can be extracted [for lymphocyte subset analysis, see FIG. 7(a)].

In this case, the process branches to Sr9, but if the sample is abnormal and the inflection point cannot be extracted, the process branches to Sr7 to notify the operator of the abnormality. This notification is performed by display on the CRT 23 or by voice. Then, manual correction of the fraction is performed by the operator (Sr8), and the process proceeds to Sr1.

In Sr1, ■. Inflection points p1, pz from the histogram
, extract ps, ! ,. Data for which is between Pl and p2 are selected and collected from all measurement data for that sample. Figures 5(a) and 5(b) show the selected and collected data! ,. and ■. The histogram is shown. Furthermore, ■ shown in FIG. 5(C).

From the histogram, inflection points are extracted, and fraction B is finally determined based on these inflection points (STIO1 6th
(see also figure).

In the continuation <5TII, calculations for the final fraction are performed on the data in the fraction determined in 5TIO. To explain this calculation with reference to Figure 4 (b), first,
From the data in the fraction determined by 5TIO, the highest carbon number was 4.
0 point 9 is extracted (ST111). Then, from this point q, a straight line l is formed radially at every constant angle θ (ST
112). For example, in the case of lymphocyte subset analysis, as shown in Figure 1, the highest numerical value point q is extracted in fraction B, and straight lines are drawn radially every 30 degrees from this point q!
, ~1+t. Note that the angle is not limited to 30°, and it is also possible to form straight lines at non-equal angles.

Next, create a histogram on each straight line i (STI
13), FIGS. 2(a), 2(b), and 2(C) are schematic diagrams illustrating examples of conohistoclam. Next, it is determined whether an inflection point can be extracted from the histogram obtained in 5T113, and if this determination is YES, the process branches to 5T115, and if No, the process branches to 5T116 (ST11
4). At 5T115, it is further determined whether there is one inflection point. If YES, the process branches to 5T117; if NO (if there are two or more inflection points), the process branches to 5T11B.

FIG. 2(a) shows a case where there is no inflection point in the histogram. In this case, proceed to 5T116 and threshold value n
Let the point where the two intersect with each other be the boundary point r. Figure 2(b) shows a case where there is one inflection point X, but in this case,
The process advances to 5T117, and that one inflection point is determined as the boundary point r. Figure 2 (C) shows a case where there are two inflection points X, , X, but in this case, proceed to 5T118 and set the inflection point Let it be r.

In 5T119, it is determined whether the boundary points r have been determined for all the straight lines l, and if this determination is NO,
The process advances to 5T113 to create a histogram for the next straight line, and if YES, returns to the main routine of FIG. 4(a) and proceeds to the process of 5TI2. In addition, FIG. 1 shows each straight line IAI"' obtained by the final fraction calculation.
Boundary points r, ~r1□ for I11 are shown.

In 5T12, the area obtained by connecting the boundary points r obtained in 5TII with a straight line is determined as the final fraction.

In the case of FIG. 1, the area B° obtained by connecting the boundary points r1 to r+z with straight lines is determined as the final fraction. This final fraction B' has a shape that is more compatible with the distribution of lymphocytes than fraction B obtained by 5TIO.

Furthermore, the processing time of 5TII, 12 can be shorter than that of the contour tracking method.

Data belonging to the final fraction determined in 5T12 is further subjected to fluorescence characteristic analysis (ST13). In this fluorescence characteristic analysis, for example, histograms are created for each of the green fluorescence intensity I9 and the red fluorescence intensity ■, and the positive rate is calculated. The results of this fluorescence characteristic analysis are C
It is displayed on the RT 23 (ST14) and printed out from the printer 24 (ST15).

At 5T16, it is determined whether there is a sample to be measured. If this determination is YES, the process returns to Sr1 and the next sample is measured; if the determination is NO, the measurement is ended.

In the above explanation, the analysis of lymphocyte subsets is taken as an example, but the present invention is not limited to this. Furthermore, the parameters used are not limited to the four types of forward scattered light intensity, 90° scattered light intensity, green fluorescence intensity, and red fluorescence intensity. It is also applicable to cell analyzers that use the window method.
In this case, determine the final fraction within the window.

(g) As described in detail of the invention, the cell analyzer of the present invention is capable of determining parameters related to the fractionation of the cell population regarding the cytooptical information of the target cell population collected by the cytooptical information collection means. Highest frequency chain point extraction means for extracting the highest frequency chain point extraction means on the cytogram; and straight line formation for forming a straight line radially from the highest frequency chain point extraction means on the cytogram. From the means and histograms on each straight line formed by this straight line forming means,
A boundary point extraction means for extracting boundary points and a region formed by connecting the boundary points extracted by this boundary point extraction means are defined as a final fraction, and based on this final fraction, cytooptical information of the target cell population is obtained. Since it is equipped with a second cell optical information collection means, it has the advantage of being able to determine a fraction that is more suitable for the target cell population in a short processing time, and improving analysis accuracy. There is.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the main part of a cytogram for explaining the final fraction calculation of a cell analyzer according to an embodiment of the present invention, FIG. 2(a), FIG. 2(b), and FIG. (C) is a schematic diagram of a histogram explaining the final fraction calculation of the same cell analyzer, FIG. 3 is a block diagram explaining the configuration of the same cell analyzer, and FIG. Figure 4 (b) is a flow diagram explaining the overall operation of the analyzer, and Figure 5 (a) is a flow diagram explaining the final fraction calculation of the cell analyzer.
5(b) is a histogram of 90° scattered light intensity and forward scattered light intensity, respectively, to explain the fraction extraction calculation of the same cell analyzer, and FIG. 6 is a histogram of forward scattered light intensity, 90° scattered light intensity.
°Schematic diagram showing an example of a cytogram of scattered light intensity, No. 7
Figures (a) and 7) are diagrams showing examples of histograms of the 90° scattered light intensity and the forward scattered light intensity, respectively. 10; Flow cell, 14: Laser, 15a ・15
b-15cm 15d: Photodetector, 21? MPU
23: CRT. 24: Printer. Figure 1 Figure 2 (a) Figure (F3) Figure (b) Figure 1 Lily I90 (ch) - LO (C/1) - 1φ (a) Procedural amendment (voluntary) Circular) 1988 October 5th

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 detects cell optical information consisting of multiple parameters for each cell irradiated with this light beam. a cell optical information detection means for detecting cell optical information; a cell population fractionating means for fractionating a cell population in the cell suspension based on one or more parameters obtained by the cell optical information detection means; Based on one or more fractions among the fractions obtained by the population fractionation means,
a cell optical information collecting means for collecting cell optical information of a target cell population; a cell optical information detected by the cell optical information detecting means and a target cell collected by the cell optical information collecting means; In a cell analysis device comprising a cell optical information processing means for processing cell optical information of a population, and an output means for outputting a processing result of the cell optical information processing means, the cell optical information collecting means comprises: a highest numerical point extraction means for extracting the highest numerical value point on the cytogram of the parameter related to the fractionation of the cell population, with respect to the collected cyto-optical information of the target cell population; Straight line forming means for forming a straight line radially from the highest frequency value point extracted by the frequency value point extraction means; Boundary points for extracting boundary points from a histogram on each straight line formed by the straight line forming means an extraction means; and a second cytooptical device that defines a region formed by connecting the boundary points extracted by the boundary point extraction means as a final fraction, and collects cytooptical information of a target cell population based on this final fraction. What is claimed is: 1. A cell analysis device characterized by comprising: information collection means.