JPH03229152A - Cell analyzing apparatus - Google Patents

Abstract

PURPOSE:To make analyses automatically with high efficiency by using two-dimensional smoothing processing for processing of the information on cell light. CONSTITUTION:A sample is sucked by a sample suction tube 4 from a sample container 3 and is sent to a flow cell 10 when a measurement is started. Each one of the cells flowing in one array on the central axis of a flow channel 10a is irradiated with a laser beam l and the intensity of scattering light is measured by photodetectors 15a to 15d. The photodetection signals are sent via a signal processing circuit 20 to a main processor unit MPU 22. The MPU 22 computes the information on the cell light from the output of the circuit 20 by a two-dimensional smoothing method upon ending of the measurement. The adequate autotrigger computation is executed even when the number of data is little or when the extraction of the minimal point is difficult. The automatic measurement with the higher efficiency is thus possible and the higher reliability is obtd.

Description

Detailed Description of the Invention (a) Industrial Application Field The present invention relates to a cell analysis device that applies flow cytometry. Specifically, in the process of processing optical information of cells, two-dimensional smoothing of data is performed. This invention relates to a cell analysis device that performs.

(b) Conventional technology Flow cytometry involves passing cells or particles labeled, for example, with a fluorescent dye, into a fine liquid stream, and using a hydrodynamic focusing effect, a laser beam is focused on each cell flowing in a line. It irradiates light and instantly measures the intensity of scattered light and fluorescence generated by cells, that is, cell light information, and analyzes cells. Flow cytometry has the feature of being able to analyze large amounts of cells at high speed and with high precision, and is widely used for everything from clinical tests to research.

The cell analysis device that applies the above-mentioned flow cytometry includes a flow cell for forming a thin liquid flow, a light beam #I (for example, a laser) that irradiates the cells flowing inside the flow cell, and a light beam that irradiates the cells. A known device is equipped with a photodetector that detects cell light information from cells that have been exposed to cells and converts it into an electrical signal, and a computer that performs analysis processing of the cell light information converted into the electrical signal.

This conventional cell analysis device flows a cell suspension (sample) labeled 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, blood is reacted with a monoclonal antibody labeled with a fluorescent dye and hemolyzed, and the sample is used. contains monocytes and granulocytes, and from the cell light information obtained by measurement,
It becomes necessary to select those regarding lymphocytes.

Therefore, a cell analyzer equipped with a so-called auto-trigger function, which automatically selects and collects the cell light information of the necessary cell population without relying on the subjectivity of the operator,
There is a patent application No. 62-22884 related to the applicant's earlier application. This cell analyzer fractionates cell light information based on ① or 2 or more parameters, and the resulting 1/2 fraction is
Alternatively, two or more regions are used as analysis regions to collect cell optical information of a target cell population.

For example, in the case of lymphocyte analysis, Figure 10(a)(b)
), the 90° scattered light intensity ■,. , forward scattered light intensity■. Create histograms for each.

■,. From the histogram, the minimum points P+, fez,
P:1 is the minimum point P4, p in the histogram of Io
S is extracted.

Due to these minimum points p1 to p, ■9°-■. The cytogram is fractionated as shown by the broken line in FIG. In FIG. 8, b shows the distribution of lymphocytes, C shows the distribution of monocytes, and d shows the distribution of granulocytes. Further, a shows 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. Fraction B, indicated by the solid line in FIG. 8, contains the distribution of lymphocytes, so this fraction B is used as the analysis area and cell light information is collected. The fluorescence characteristics of the collected cell light information are further analyzed and processing such as determining the positive rate is performed.

Summer, with the above cell analyzer. - On the I0 cytogram, a rectangular region B is set as an analysis region, but in such a rectangular analysis region, unnecessary information is still included in the collected cell light information. Therefore, the applicant of the present invention has already filed Japanese Patent Application Nos. 1983-193033 and 1983-193072 as a cell analyzer that can set an analysis area more suitable for the analysis of a target cell population.

The cell analyzer of Japanese Patent Application No. 193033/1983 is capable of detecting infraction parallel to the I9 axis within the fraction B, as shown in FIG. 9(a). , create a histogram on each line L, extract the minimum points or points that meet a predetermined threshold from this histogram, and connect these extracted points to create an area obtained as the analysis area B''. This method extracts cell light information as follows.

On the other hand, the cell analysis device of patent application No. 193072/1983 is
As shown in FIG. 9 (b), the highest numerical value point q is determined in the fraction B, and a line ! is drawn radially from this point q. , create a histogram for each line lJ, extract the minimum points or points that reach a predetermined threshold value from each histogram, and define the area obtained by connecting these extracted points as the analysis area B.
″′.

With these two cell analyzers, it is possible to set an analysis region closer to the distribution shape of the target cell population, thereby improving the reliability of the measurement results.

Furthermore, in Japanese Patent Application No. 63-320915, in order to unify the number of cells belonging to the set analysis area, as shown in Figure 7, cell light information is collected at slightly more points (n'), and Cut off unnecessary data using methods (a), et al. or (C), and calculate the number of cells to nl. A method is used to unify the With this cell analyzer, the objectivity of data is greatly improved by unifying the number of cells, and analysis can be automated and made more efficient.

(c) Problems to be Solved by the Invention The cell analyzer equipped with the auto-trigger function described above certainly has advantages such as automation of measurements and objective data processing. However, if the number of cells is small or the distribution of cells is uneven or uneven, ■. −■.

Histogram of forward scattered light intensity I0 from cytogram,
Alternatively, a histogram of 90'' scattered light intensity (2) cannot be created, or even if a histogram can be created, so-called "missing parts" may occur in the quantization of consecutive numbers. In this case, it becomes impossible to extract minimum points or points that meet a predetermined threshold from each histogram, which prevents auto-trigger calculations from being performed properly, hinders measurement automation, and impairs data reliability. Sometimes.

This invention has been made in view of the above, and provides a cell analysis device that can further automate and improve the efficiency of analysis by using two-dimensional smoothing processing in the processing means for cell optical information. intended to provide.

(d) Means for solving the problems In order to solve the above problems, the cell analysis device of the present invention includes:
It has the configuration listed in section 1-1 below.

i: A flow cell through which a cell suspension is flowed, ii: A light source that irradiates a light beam to the cells flowing within this flow cell, and iii: Cell light information consisting of multiple parameters for each cell irradiated with this light beam. iv: an analysis region setting means for setting an analysis region based on one or more parameters obtained by this cell light information detection means; . . .: this analysis region setting means a cell photonic information collecting means for collecting cell photonic information of a target cell population within the analysis area set by; (2): cell photonic information detected by the cell photonic information detecting means and the cell photonic information collecting means; viIi: a cell light information processing means for processing the cell light information of the target cell population collected by the cell light information processing means; The information processing means includes a two-dimensional smoothing means for two-dimensionally smoothing the cell light information detected by the cell light information detection means and the cell light information of the target cell population collected by the cell light information collecting means. It is characterized by this.

(E) In the cell analyzer of the second invention, when processing cell optical information, two-dimensional smoothing processing is applied to the cellular optical information, so that a histogram cannot be created or the histogram has "missing parts". Avoid situations that arise.

Therefore, even when the number of cells is small or when the distribution of cells is uneven or uneven (for example, the distribution of granulocytes in blood), auto-trigger calculation can be performed appropriately, promoting automation and efficiency of measurement. At the same time, it is possible to improve its reliability.

(F) Embodiment An embodiment of the present invention will be described below with reference to FIGS. 1 to 6.

FIG. 3 is a diagram showing the configuration of an example cell analysis device.

2 is an autosampler, and a plurality of sample containers 3, . . . , 3 are loaded in its sample rank 2a. 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-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 IO 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, 15b1.15
c, 15d are arranged. A laser beam l from the laser 14 is irradiated onto cells (or particles) flowing through the flow channel lOa. Signal light is generated from this cell, and the signal light in the forward direction 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 is transmitted through the lens 16b.
The light is focused. A portion of this signal light is reflected by a dichroic mirror 18a and enters a photodetector 15b for detecting 90'' scattered light.A portion of the signal light transmitted through the dichroic mirror 18a is further reflected by another dichroic mirror 18a. It is reflected by the mirror 18b and passes through the filter 19.
The light passes through the light a and is received by the green fluorescence photodetector 15c.

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

The light reception signals of the photodetectors 15a, 15b, 15c, and 15d are amplified and noise removed by a signal processing circuit section 20, and then converted into digital signals by an analog/digital (A/D) converter 21. It is taken into MPtJ22.

The MPU 22 has functions that are broadly divided into auto-trigger related functions, cell number counting/unification functions, and other functions. For example, if the method of Japanese Patent Application No. 63-193033 is applied, the function related to the auto trigger is the forward scattered light intensity [. , a function to create a cytogram and each histogram for 900 scattered light intensity [qa, a function to extract minimum points from these histograms and divide cell light information into two parts, a function to create a cytogram for the entire cytogram or two parts within both parts. It has a function to perform dimensional smoothing, a function to determine the final two parts within the two parts including the target cell population, etc.

Functions related to cell number counting and unification include a function to store the final fraction from the previous measurement, a function to count the cell number using the final fraction from the previous measurement during the current measurement, and a function to count the cell number using the final fraction from the previous measurement. It has a function of adjusting the number of cells in the cell to a specified number n1°.

Other functions include a function to perform fluorescence analysis on optical information of a target cell population with a uniform number of cells, and a function to control the sample pump 7, liquid pump 9, and autosampler 2.

A floppy disk drive 23, a keyboard 24, a CRT 25, and a printer 26 are connected to the MPU 22. The floppy disk drive 23 is for storing measurement conditions (protocols), measurement data, etc. on a floppy disk. The keyboard 24 is used to input protocol selection/setting or other commands to the MPU 22 . The CRT 25 is for monitoring measurements, and the printer 26 is for printing out processing results of the MPU 22, such as cytograms and histograms.

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
Fluorescein isothiocyanate (FI) was added to the patient's blood.
It is obtained by reacting 0KT4 monoclonal antibody labeled with TC) and 0KT8 monoclonal antibody labeled with phyconilithrin (PE), followed by hemolysis treatment.

When the cell analyzer is powered on, the ROM (not shown)
The program is read into the MPU 22 and the system starts up [Step 7 (hereinafter referred to as ST) l, see Figure 2]
. Next, a protocol suitable for the sample to be measured is selected and set. This protocol includes a photodetector 15aS 1
The contents include measurement conditions such as detection gains and correction calculations of 5b, 15c, and 15d. 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.

Next, in Sr1, it is determined whether a command to perform an auto trigger has been input from the keyboard 24 or not. If this determination is YES, the process branches to Sr1, and if this determination is No, the process branches to Sr1. Further, in Sr1, the MPU 22 determines whether or not a command has been input indicating that the current measurement is to be performed according to a new protocol different from the previous measurement.

If this determination is No, the process branches to Sr8, and if this determination is YES, the process branches to Sr1.

The explanation will now proceed assuming that Sr1 or branched from Sr1 to Sr1. In Sr1, the operator selects window B0 from the keyboard 24 to 1. . −1° is set on the cytogram [see Fig. 5(a)].

Once window B0 is set, measurement starts (S
r1). At Sr1, the sample rank 2a is driven, and the sample container 3 containing the sample to be measured first is positioned directly below the sample suction tube 4.

Then, the three-way valve 5 is switched to communicate the sample suction tube 4 and the sample liquid feeding tube 4, 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 suction tube 4 is moved to the suction side. is sucked into the sample liquid feeding tube 6a.

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 sending pump 9 is driven to the liquid sending side, and the sheath liquid is sent to the flow cell IO. A sheath flow is formed within the flow channel 10a, and cells flow in a line on the central axis of the flow channel 10a due to the hydrodynamic focusing effect. Each cell is irradiated with a laser beam i, with forward scattered light intensity t, and 90° scattered light intensity I. ,
The green fluorescence intensity I9 and the red fluorescence intensity ■ are measured respectively. These cell light information are sequentially stored on a floppy disk or the like as necessary.

During the measurement, the number of cells belonging to the window B0 is counted. In Sr1, it is determined whether this cell number has reached a predetermined number, and if YES, the process goes to Sr1.
In this case, repeat Sr1. The predetermined value n° is the predetermined number of cells n. Larger than n. It is assumed to be +α.

In Sr1, the measurement is finished and calculations are performed on the cell light information within the window 0. First, the cell light information within the window is calculated using a two-dimensional smoothing method. As the two-dimensional smoothing, for example, a method of calculating as a weighted distance function as shown in FIGS. 4(a) and 4(b) or a method of separating the background as shown in FIG. 4(C) is used. After this, the above-mentioned patent application No. 193033/1983 [see Figure 9(a)]
The final fraction B0° is determined according to the procedure. In addition, the fourth
In the figure, when N=5 [Fig. 4 et al.], 9 [Fig. 4 (a)]
[case]], however, masks with N=13.25 may be provided. Furthermore, when averaging the value at the center of the mask, weighted averaging or moving averaging is performed.

At this time, the number of cells n in the final fraction B01 is smaller than the predetermined number of cells n, but since the count was made with a margin of α from the beginning, n. It will never fall below.

In 5T12, cell number unification processing is performed. This is the cell light information (number of cells n) in the final fraction B0, as shown in Figure 7.
Among them, nl. This is done by cutting off the part that exceeds . The parts to be cut off include cutting off the last part as shown in Figure 7 (a), cutting off the first part as shown in (ra), or cutting off the first and last parts as shown in (C). There is a method.

Fluorescence characteristic analysis is further performed on the data in which the number of cells has been unified at 5T12 (ST13).

In this fluorescence characteristic analysis, for example, histograms are created for green fluorescence intensity 9 and red fluorescence intensity 1, respectively.
The positivity rate will be calculated. The results of this fluorescence characteristic analysis are displayed on the CRT 25 (ST14) and printed out from the printer 26 (ST15).

At 5T16, it is determined whether there are any more samples to be measured. When this determination is YES, the process returns to STI, and when this determination is NO, the analysis ends.

Now, return to STI and use the same protocol as the previous measurement.
If auto-triggering is to be performed, the process proceeds to Sr1 via Sr2 and Sr1. In Sr1, the final fraction B0'' obtained in the previous measurement is again set as a window on the cytogram [see Figure 3 (b)].
. Then, measurement is started in exactly the same manner as Sr5, and the cells entering the final fraction B are counted, and it is determined whether the number has reached a predetermined number n (STIO).

When the determination at 5TIO becomes YES, the process advances to 5TII to end the measurement and perform auto-trigger calculation. In this auto-trigger calculation, we first obtained 190,! . The data is subjected to a two-dimensional smoothing operation as before (STI 11, see Figure 1). Next, 1 is two-dimensionally smoothed. ,■. Per data, Iqo I.

Create a cytogram, 19°, I0 histogram (
ST112). From these histograms, the minimum point P+-
It is determined whether Ps can be extracted (ST113). If this judgment is YES, 5TI14, if NO,
5T115 respectively.

In 5T114, the minimum points 21 to p5 are extracted from the histogram [see Fig. 10 (a) and (b)], and based on these, fraction B1 is set [see Fig. 5, etc.]], and the data in this fraction B+ are Collect. Then, this fraction B is subjected to the treatment described in Japanese Patent Application No. 193033/1988 [see FIG. 9(a)] to determine the final fraction B1° (ST117).

On the other hand, with 5T115, the minimum point cannot be extracted.
The operator is notified by displaying it on the CRT 25 or the like.

Then, manual setting of fraction B1 is performed by the operator (
ST116). Regarding the fraction B1 set in this way, the final fraction B1° is determined by the process described in Japanese Patent Application No. 193033/1988 [see FIG. 9(a)] (ST117).

In 5T11B, it is determined whether or not to end the two-part determination. For example, if there is an input from the keyboard 24 to redo the final fraction Bl', this determination will be No and the 5T will be repeated again.
Return to 116. Otherwise, this judgment is YES
Then, the process returns to the main routine shown in FIG.

Alternatively, in the auto-trigger calculation of 5TII, as in the case of Sr7, extract the minimum point P+-Ps, set the fraction B, and then calculate ■ within this percent B1. , to data may be subjected to two-dimensional smoothing processing to determine the final fraction B, l.

In both cases, the number of cells is counted from the immediately preceding final fraction B, so that the number of cells in the final fraction B,'', l
is different from n゛, but is never less than neo.

Therefore, in this case as well, perform cell number unification processing as before (
ST12). Then, the processes of 5T13 to 5T15 are performed.

FIGS. 6(a) and 6(b) show a state in which the final fraction B is determined by performing autotrigger calculation on granulocytes.
[. In the cytograms, FIG. 6(a) shows the case where two-dimensional smoothing processing was not performed, and FIG. 6(a) shows the case where two-dimensional smoothing processing was performed. In FIG. 6(a), the final fractions B and 1 are disordered, indicating that there is a high possibility that the final fraction Bl' is not set appropriately. In contrast, FIG. 6(b) shows that the final fraction 8
It can be confirmed that the boundary of 1' is smooth and the final fraction B1° is appropriately set.

In this way, the cell analyzer of this example performs two-dimensional smoothing of the data and performs auto-trigger calculations within a fraction or the entire 9°-I0 cytogram. Even when extraction is difficult, auto-trigger calculation can be performed appropriately, promoting automation of measurement and improving reliability.

Although the above examples describe lymphocytes and granulocytes, the present invention is applicable to the analysis of a wide range of samples such as white blood cells in general such as monocytes, red blood cells, and cultured cells.

In addition, as an auto trigger method, patent application No. 1983-19
Not limited to the one of No. 3033, but the patent application No. 63-193
072, that of Japanese Patent Application No. 62-22884, and other contour tracking methods can be modified as appropriate.

(g) As described in detail of the invention, the cell analyzer of the present invention is characterized in that the cell light information processing means collects the cell light information detected by the cell light information detection means and the cell light information collection group. The present invention is characterized by comprising two-dimensional smoothing means for two-dimensionally smoothing cell light information of a target cell population. Therefore, even when there are "missing data", noisy samples, or special samples, auto-trigger calculation can be performed appropriately, which has the advantage of promoting measurement automation and improving reliability. have.

[Brief explanation of drawings]

FIG. 1 is a flowchart explaining the auto-trigger operation of a cell analyzer according to an embodiment of the present invention, FIG. 2 is a flowchart explaining the overall operation of the cell analyzer, and FIG. A block diagram explaining the configuration of the cell analyzer, Fig. 4 (a)
), FIG. 4(b), and FIG. 4(C) are diagrams explaining the two-dimensional smoothing process of the same cell analyzer, and FIG. 5(a), respectively.
) and FIG. 5(b) are cytograms of 90° scattered light intensity-forward scattered light intensity, respectively, and FIG. 6(a) and FIG. 6(
b) is a cytogram of 90° scattered light intensity vs. forward scattered light intensity, showing a comparison of two-dimensional smoothing of granulocytes with and without two-dimensional smoothing in the same cell analyzer, and FIG. Diagrams for explaining the unification of cell numbers in conventional cell analyzers, Figures 8, 9 (a) and 9 (b)
10(a) and 10(b) are cytograms of 90° scattered light intensity-forward scattered light intensity to explain the auto-trigger of a conventional cell analyzer, respectively. It is a histogram of 90 degree scattered light intensity and forward scattered light intensity for explaining the auto-trigger of the device. lO: Flow cell, 14: Laser, 15a/15b
・15cm15d: Photodetector, 22: MPU,
25: CRT, 26: Printer.

Claims (1)

[Claims] (1) A flow cell through which a cell suspension is passed, a light source that irradiates a light beam onto cells flowing within this flow cell, and detects cell light information consisting of multiple parameters for each cell irradiated with this light beam. A cell light information detection means; an analysis region setting means for setting an analysis region based on one or more parameters obtained by the cell light information detection means; , a cell light information collecting means for collecting cell light information of a target cell population, and cell light information detected by the cell light information detection means and cell light information of the target cell population collected by the cell light information collecting means. In a cell analyzer comprising a cell light information processing means for processing information and an output means for outputting a processing result of the cell light information processing means, the cell light information processing means is a cell light information detecting means. A cell analysis device comprising a two-dimensional smoothing means for two-dimensionally smoothing detected cell light information and cell light information of a target cell population collected by the cell light information collecting means.