JPH0443942A - Cell analyzer - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

Detailed Description of the Invention (a) Industrial Application Field The present invention relates to a cell analysis device applying flow cytometry, and more specifically, relates to the collection of cell light information in the process of processing cell light information. .

(b) Conventional technology Flow cytometry involves flowing cells or particles labeled, for example, with a fluorescent dye, into a thin 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 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 device is known that includes a photodetector that detects cellular optical information from cells and converts it into an electrical signal, and a computer that performs analysis processing of the cellular optical information converted into the electrical signal.

This cell analyzer can be applied to the analysis of various cells such as red blood cells, white blood cells, and cultured cells.

For example, in the case of blood lymphocyte subset analysis,
A monoclonal antibody labeled with a fluorescent dye is reacted with human blood, and then this blood is hemolyzed and used as a sample.

The sample cells flowing inside the flow cell are irradiated with a laser beam, and four parameters are determined: forward scattered light intensity 1
．． ．． 90'' scattered light intensity T, ., green fluorescence intensity I9, and red fluorescence intensity ■ are detected by the detector, respectively, and cell light information (hereinafter sometimes simply referred to as data) is obtained.In the above sample, In addition to lymphocytes, monocytes, granulocytes, etc. are included, and the obtained data will include data on monocytes, granulocytes, etc. in addition to data on lymphocytes. Therefore, it is necessary to collect data on lymphocytes by selecting them from data on other cells.

Therefore, the forward scattered light intensity 1. which is said to represent the size and internal structure of cells. ．． 90° scattered light intensity ■. A cytogram is created with the orthogonal coordinate axes as shown in FIG. 4(a). In this cytogram, b shows the distribution of lymphocytes, C shows the distribution of monocytes, and d shows the distribution of granulocytes. Note that the triangular area f is a portion removed by noise processing. On this cytogram, set a polygonal or elliptical analysis area (often called a window) B that includes, for example, the distribution of lymphocytes and belong to this analysis area (belong to the shaded area). Cell data is collected by sorting it from data on other cells. This analysis area can be set manually by the operator using a mouse or digitizer, or automatically (so-called auto trigger, for example, Japanese Patent Application No. 63-193033, Japanese Patent Application No. 63-19307).
(See No. 2).

Regarding the lymphocyte data collected in this way, the green fluorescence intensity ■9 and the red fluorescence intensity i are subjected to arithmetic processing, for example, as shown in FIG. 4(b), 1, -1. A cytogram is created and the positivity rate is calculated.

(c) Problems to be Solved by the Invention In the above-mentioned cell analyzer, samples are often prepared from human blood, but blood reflects various human pathological conditions.
■,. −■. The distribution on the cytogram also exhibits a complex pattern, and the distribution of cells with different affiliations overlaps in various ways, making it sometimes impossible to identify the target cell population on the cytogram.

For example, in the above-mentioned lymphocyte subcentrant distribution, in the case of a healthy person, the cell population is roughly divided into three as shown in Figure 4(a), and the analysis area B, which includes the lymphocyte distribution, is divided into three groups.
As described above, the fluorescence analysis is performed using the lymphocyte data belonging to this analysis region B. However, in the case of a leukemia patient, as shown in FIG. 4(C), the distribution e may be such that lymphocytes and other cells overlap, making it impossible to accurately set the analysis region B.

Therefore, there was a problem in that the positive rate of the fluorescent dye-labeled monoclonal antibodies used varied greatly.

On the other hand, there may be cases where it is desired to perform analysis on all cell populations in a sample, excluding a certain cell population.

For example, if you want to analyze the part of the above sample excluding lymphocytes, you must set the analysis area excluding the distribution of lymphocytes, as shown in Figure 4(d). Even though this area does not belong to the distribution of lymphocytes, it leaks from the analysis area, making it impossible to perform accurate analysis.

The present invention was made in view of the above, and aims to accurately identify and selectively collect data on necessary cell populations, and to accurately analyze complex cytogram patterns.

(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 configurations listed in 1 to IX below.

i: A flow cell through which a cell suspension is flowed, ii: A light source that irradiates a light beam to cells flowing in this flow cell, and ji = 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; V: an analysis region setting means for detecting Within the set analysis area,
a first cell light information collecting means for collecting cell light information of a target cell population; vi: cell light information detected by the cell light information detecting means and information collected by the first cell light information collecting means; and vi: an output means for outputting the arithmetic processing result of the cell light information arithmetic processing means; vm: the above-mentioned analysis. a second cell light information collecting means that collects cell light information outside the analysis area set by the area setting means in a complementary manner; The present invention is characterized in that the cell light information collected by the information collecting means or the second cell light information collecting means is selected and subjected to arithmetic processing.

(E) Function The cell analyzer of the present invention identifies a target cell population when collecting cell optical information by identifying it in a subset manner or in a complementary manner. It is possible to more accurately identify the target cell population on the cytogram and improve the accuracy of analysis, especially the accuracy of determining the positive rate.

(F) Embodiment An embodiment of the present invention will be described below with reference to FIGS. 1 to 3.

FIG. 2 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 rank 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 boats 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 an 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. and be swept away.

The liquid flowing out from the flow cell IO 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, photodetectors (cell optical information detection means) 15a, 15b, 15c,
15d is provided. Laser beam from laser 14!
is irradiated onto cells (or particles) flowing through the flow channel 10a. Signal light is generated from this cell, and the signal light in the forward direction is transmitted to the lens 16 as forward scattered light.
The light is focused on point a and enters the photodetector 15a. 17 is
This is a beam protection device 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 the microink 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 18b, and a 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 5d. Note that, for example, the laser 14 includes an argon laser, a helium neon laser, and a photodetector 15a for forward scattered light.
includes a photodiode and other photodetectors 15b and 15c.
, 15d, a photomultiplier tube is applied.

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 the MPU 22.

The MPU 22 has a function of creating a scattered light intensity ■9°-10 cytogram. −■. A function to set a window on the cytogram, a function to collect data within or outside the window, a function to perform fluorescence characteristic analysis on the collected data, and other controls for the sample pump 7, liquid pump 9, and autosampler 2. It has functions such as

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 2G is for printing out processing results of the MPU 22, such as cytograms and histograms. Although not shown, input means such as a mouse is also connected.

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 inthiocyanate (F) is added to the patient's blood.
It is obtained by reacting 0KT4 monoclonal nano-G antibody labeled with ITC) 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 (hereinafter referred to as ST) 1, see Figure 1).
. Next, a protocol suitable for the sample to be measured is selected and set. This protocol includes photodetectors 15a, 15
The content includes measurement conditions such as detection gains and correction calculations for the components b, 15c, and 15d. This is because the sample processing method differs depending on the measurement purpose. For example, the monoclonal antibodies used for each treatment have different reactivity with cells, so the photodetectors 15a, 15b, 15c, 15d
It is necessary to change the detection gain, and since the binding mode between each monoclonal antibody and fluorescent dye is different, the correction calculation also needs to be changed accordingly.

In ST2, a sample is passed through the flow cell 10 and a measurement is performed. First, 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. The three-way valve 5 is connected to the sample suction 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 is sucked into the sample liquid supply 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 feeding pump 9 is driven to the liquid feeding side, and the sheath liquid is transferred to the flow cell 10.
sent to. Within the flow channel 10a, a sheath flow is formed and cells flow in a line on the central axis of the flow channel 10a due to the hydrodynamic focusing effect. Laser beam on each of these cells! is irradiated, and the forward scattered light intensity ■. , 90° scattered light intensity■,. ,
The green fluorescence intensity I9 and the red fluorescence intensity ■1 are each measured (.These cell light information are sequentially stored on a floppy disk or the like.

The MPU 22 calculates the scattered light intensity I, based on the measured data. =■. A cytogram is created (S73). This ■. -10 cytograms are displayed on CRT25,
The operator uses a mouse or the like to set a window on the 190 to sacytogram (Sr4). Note that the setting of window B may be configured to be performed automatically (auto trigger).

In Sr1, it is determined whether window B is to be reset. This determination may be made by the operator by looking at the display on the CRT 25, or by checking the inside of the window. ,
Create an I0 histogram and record its peak position, average value,
The standard deviation and coefficient of variation may be calculated and compared with a reference value to automatically determine whether or not to perform resetting. If this judgment is YES, go to Sr6, if NO, go to 5T.
Each branches to IO.

From Sr6 to Sr1, window resetting processing is performed by so-called nesting.

For example, in the case of a leukemia patient, ■. -! . As the cytogram shows in Figure 3(b), there are times when the lymphocyte distribution overlaps with the distribution of other cells, and in such cases, window B is set to surround the overlapping distribution e (
Sr4). In such a case, the determination of Sr1 is YES.
Then, the process branches to Sr6.

In Sr6, data on cells belonging to window B are collected, and based on these data, fluorescence intensity 1. -1. A cytogram is created [see Figure 3 (C)]. On this cytogram, a window C is set to surround distribution i (Sr7).

Furthermore, the MPU 22 collects data belonging to outside this window C (shaded area), and again creates a scattered light intensity 190 10 cytogram for the collected data [see Fig. 4 (1)]. I. - ■. On the cytogram, the overlapping portion is removed and the distribution of lymphocytes appears. Therefore, the window B° that accurately includes the distribution of lymphocytes can be reset.

At 5TIO, the MPU 22 collects data within this reset window (indicated by diagonal lines) and again sets the fluorescence intensity to 1. -1. Cytogram (or ■9
Create a histogram (■, histogram) and calculate the positive rate, etc. If the Sr1 judgment is NO, directly 5TIO
If the process branches to , data is collected within window B set in Sr4, and the same processing is performed.

In addition, if you want to perform fluorescence characteristic analysis on cells other than lymphocytes, as shown in Figure 3 (a), collect data outside window B (shown with diagonal lines) and perform fluorescence characteristic analysis. conduct.

With 5TII, the results of fluorescence characteristic analysis with 5TIO are CR
The result is displayed at T25, and further printed out from the printer 26 at 5T12.

At 5T13, 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.

Although this example is explained using the distribution of lymphocytes as an example, 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.

(g) As described in detail of the invention, the cell analysis device of the present invention includes a second cell light information collection system that collects cell light information outside the analysis region set by the analysis region setting means in a complementary manner. The cell light information calculation processing means selects and processes the cell light information collected by the first cell light information collection means or the second cell light information collection means. Because of this, it has the advantage of being able to accurately identify and collect data on the necessary cell means and to analyze complex cytodulum patterns with greater precision.

[Brief explanation of drawings]

FIG. 1 is a flow diagram explaining the operation of a cell analyzer according to an embodiment of the present invention, FIG. 2 is a block diagram explaining the configuration of the cell analyzer, and FIGS. Figure (c)
, Fig. 3(C) and Fig. 3(d) are cytograms explaining the relationship between the window and the data collection area in the same cell analyzer, Fig. 4(a) and Fig. 4(d), respectively. figure~
), FIG. 4(C), and FIG. 4(d) are cytograms each illustrating the relationship between the window and the area for collecting data in a conventional cell analyzer. 10: Flow cell, 14: Laser, 15a15b1
5c/15d: Photodetector, 22: MPU, 25
: CRT, 26: Printer. Figure (a) ls. Figure (b) Figure (a) ls. Figure (b) Figure (C) Figure (d) Figure (C) Is. Figure (d)

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 first cell light information collecting means for collecting cell light information of a target cell population; and a cell light information detected by the cell light information detecting means and the cell light information collected by the first cell light information collecting means. A cell analyzer comprising a cell light information arithmetic processing means for arithmetic processing of cell light information of a target cell population, and an output means for outputting the arithmetic processing result of the cell light information arithmetic processing means, the analysis area setting means a second cell light information collecting means for collecting cell light information outside the analysis area set in a complementary set; A cell analysis device characterized in that the cell light information collected by the cell light information collection means of item 2 is selected and subjected to arithmetic processing.