JPH0240557A - Analyzing apparatus of cell - Google Patents

Abstract

PURPOSE:To improve the reliability of a result of measurement and to measure a number of samples efficiently by inputting optimum conditions of measurement beforehand for each of a plurality of treating methods applied to the samples and by conducting switching over to the optimum conditions of measurement in the sequence of measurement of the samples. CONSTITUTION:Samples of blood collected from one person, for instance, which are treated by five different treating methods alpha to epsilon, are put in five different sample vessels 3 and set on a sample rack 2a. First, optimum measuring conditions (detection gains of photodetectors 15a to 15d and the contents of corrective computations) for each of the samples subjected to the treatments alphato epsilon are inputted to MPU 21 from a keyboard 22. Measurement is conducted in such a manner that samples 3 set in an autosampler 2 are measured in such specified sequence as a sample (5-alpha) of a fifth person treated by the treatment alpha, a sample (3-gamma) of a third person treated by the treatment gamma,..., for instance. In accordance with this sequence of measurement, measuring conditions for the treatment alpha, measuring conditions for the treatment gamma,... are selected and set automatically, and measured data are processed.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application This invention relates to a cell analyzer applying flow cytometry, and more specifically, to optimal setting of measurement conditions.

(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, each cell flowing in a line is irradiated with a beam of light such as a laser, and the intensity of scattered light and fluorescent yellow generated by the cells, in other words, the cell optical information, is instantaneously measured. , to analyze 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 beam a (e.g. laser) that irradiates the cells flowing within the flow cell, and a light beam that irradiates the cells flowing through the flow cell. A known device is equipped with a photodetector that detects cell optical information from cells and converts it into an electrical signal, and a computer that performs analysis processing of the cell optical information converted into the electrical signal.

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,
OKT 4-E-/clonal antibody and phyconilithrin (PE: red fluorescence) labeling (fluorescein isothiocyanate) (FITC: green fluorescence) labeling
T8 monoclonal antibody] is used as a sample.

The sample cells flowing through the flow cell are irradiated with a laser beam, and four parameters are determined: forward scattered light intensity 1
．． ．． 90° scattered light intensity I. , a green fluorescence intensity of 19, and a red fluorescence intensity of 1 are detected by a detector, respectively, to obtain cell optical information (hereinafter sometimes simply referred to as data).

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, we create a cytogram with forward scattered light intensity re, 9o° scattered light intensity■9° as the orthogonal coordinate axes, and set an analysis area called a window on this cytogram to select lymphocyte data. Methods are known (see, for example, Japanese Patent Laid-Open No. 134559/1983). In this way, the data of the selected lymphocytes are analyzed for green fluorescence intensity (19) and red fluorescence intensity (2), and the positive rate is calculated.

(c) Problems to be Solved by the Invention In recent years, in order to efficiently measure a large amount of samples, the introduction of a so-called autosampler, which automatically supplies a large number of samples, has been considered for the above-mentioned cell analyzers. . This autosampler is also preferable from the viewpoint of biohazard prevention, since direct contact between the sample and the operator can be avoided.

However, there are many factors that influence the positive rate mentioned above.
Measurement conditions differ depending on the sample processing method. For example, since the reactivity of various monoclonal antibodies with cells is different, measurements cannot be made with the same detection gain even with the same detector.Also, due to differences in the binding mode between monoclonal antibodies and fluorescent substances, it is difficult to detect fluorescence intensity. Correction calculation is required.

Therefore, it is necessary to select and set the optimal measurement conditions for each sample, but this selection and setting is largely based on empirical knowledge, and conventionally the operator performs the selection and setting work while monitoring. Ta. However, this work requires skill and labor, which hinders the improvement of the reliability of measurement results and the efficient measurement of a large number of samples. Therefore,
Even if an autosampler is introduced and only the sample supply is automated, the selection and setting of measurement conditions remains the same as before.
Improvements in measurement efficiency and reliability of measurement results cannot be expected.

This invention was made in view of the above, and aims to provide a cell analyzer that can automatically set optimal measurement conditions for each sample, improve the reliability of measurement results, and efficiently measure a large number of samples. There is.

(d) Means and operation 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 cells flowing inside the flow cell. a light source that irradiates a light beam to a cell; a cell optical information detection means that detects cell optical information consisting of one or more parameters regarding a cell irradiated with the light beam; and a cell optical information detection means that detects cell optical information consisting of one or more parameters. a cell optical information sorting means for selecting cell optical information of a target cell population from the detected cell optical information; and a cell optical information detected by the cell optical information detecting means and the cell optical information. A cell analysis device comprising a cell optical information processing means for analyzing and processing the cell optical information sorted by the sorting means, and an output means for outputting a processing result of the cell optical information processing means, comprising a plurality of cells. A sample supply means for aspirating a sample 111 times and sending the aspirated sample to the flow cell, and measurement conditions for setting optimal measurement conditions in the cell optical information processing means according to the sample supplied by the sample supply means. The present invention is characterized by comprising a setting means.

In the cell analyzer of the present invention, the sample is sequentially fed to the flow cell by the sample supply means, and measurements are performed continuously. The conditions are automatically set by the measurement condition setting means. Therefore, it is possible to efficiently measure a large number of samples while improving the reliability of measurement results.

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

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

2 is an autosampler, and its sample rack 2a (
(only one is shown) includes a plurality of sample containers 3,...
3 is loaded. 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 sample rack 2a is equipped with a shaking mechanism and a cooling mechanism (not shown), and shakes the samples at regular intervals, and also keeps the samples in the sample containers 3, . . . 4°C-1
0°C).

This autosampler 2 has five different processing methods α and β for blood collected from a patient, for example.
, γ, δ, and ε are placed in five different sample containers 3 and set. That is, five types of samples are set for the - patient, and the total number of samples is the number of patients multiplied by five.

On the other hand, the sample suction tube 4 is connected to one boat of the three-way valve 5. The other two boats with three-way valve 5 have
The sample liquid feeding tubes a and 6b are connected to each other, and 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 attached to 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 a material such as quartz glass that transmits the wavelength of the light used, and a sheath flow is formed in the internal flow channel 10a, and the cells or particles in the sample are focused by the hydrodynamic focusing effect. are flowed in a line on the central axis of the flow channel 10a.

The liquid flowing out from the flow cell 10 is guided to the waste liquid tube 2 and stored in the 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, 15b1.1
5c and 15d are provided. 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 in the internal direction ψ 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 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 guichroic 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 guichroic 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 photodetectors 15b, 1
Photomultiplier tubes are applied to 5c 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 are taken into the MPU 21. MPU21
is the intensity of forward scattered light. , 90° scattered light intensity jfT,. A function that creates a cytogram or histogram of the target cell (or particle) population and selects the data of the target cell (or particle) population.The selected data is analyzed for green fluorescence intensity (■9) and red fluorescence intensity (1) to determine the positive rate. It has a function to control the autosampler 2, the sample pump 7, and the liquid feeding pump 9.

A keyboard 22, a CRT 23, and a printer 24 are connected to the MPU 21. The keyboard 22 is used for inputting mode designation, protocol (measurement condition) selection/setting, and other commands to the MPU 21 . C
RT23 monitors the measurement and has a printer (
The output means 24 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 with reference to FIGS. 1 and 3.

When the cell analyzer is powered on, the ROM (not shown)
The program is then read into the MPU 21 and the system starts operating. Then, a mode is selected [step (hereinafter referred to as ST) 1].

This mode has three modes: automatic, semi-automatic, and manual. The description will proceed assuming that the automatic mode has been selected. In this automatic mode, the samples set in the autosampler 2 are automatically and sequentially measured in the specified order. The sample designations are: sample processed by the fifth patient's processing method α (5-α), 3
A sample treated with the processing method γ of the th patient (3-γ),
It is done as follows. Of course, it is also possible to specify that the same sample be measured twice.

Continuation < In Sr1, a protocol is selected and set for the sample to be measured. The protocol is for the photodetector 15a~
These are measurement conditions such as the detection gain of 15d and the contents of correction calculation. To select and set this protocol, the optimal numerical values for the samples subjected to each processing method α to ε are input in advance, and the protocol is automatically switched to the optimal protocol in the order in which the samples are measured.

For example, if the samples are specified as (5-α), (3-γ), etc., the protocol for processing method α, the protocol for processing method γ, etc.・The settings are switched in order.

In addition to the above, the selection and settings of this protocol can also be done by, for example,
Once the measurement of the designated sample is completed, it is possible to enter a protocol input waiting state, or input a protocol for each sample.

Alternatively, select the protocol from an external computer, etc.
It is also possible to set it.

Note that the semi-automatic mode is a mode in which measurement is performed only on one specified sample; for example, if (4-β) is specified, the fourth patient's sample with processing method βΦ is aspirated, A protocol corresponding to this processing method β is automatically selected and measurement is performed. On the other hand, in manual mode, measurements are performed only on one specified sample, but the protocol is entered manually.
This is different from semi-automatic mode.

When the Sr1 process is completed, the sample rack 2a is driven, and the sample container 3 containing the sample to be measured first is positioned below the sample suction tube dfL. Then, the three-way valve 5 is switched so that the sample suction 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 (S
r3).

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 flow cell is turned on during the season.
Sent to 0. 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 1, and the forward scattered light intensity is 2. , 90° scattered light intensity 19
0, green fluorescence intensity ■9, and red fluorescence intensity I were measured ((Sr1).

The data thus measured is processed according to a predetermined format. For example, in the analysis of white blood cells, a cytogram with a forward scattered light intensity of I0.90 and a scattered light intensity of I90 is created (see FIG. 3).

On this cytogram, 1, distribution of lymphocytes b, distribution of monocytes C, and distribution of granulocytes d appear. Note that a is the distribution of a blush film component called debris, which is usually removed at the noise processing stage. Of course, the above format is not limited to leukocyte analysis.

In Sr1, analysis GW regions B, C, and D are set for lymphocyte distribution b, monocyte distribution C1, and granulocyte distribution d, and the average intensity, standard deviation (SD), coefficient of variation (CV)
Calculate and compare this with the pre-input reference value,
Determine whether the data is abnormal. When this determination is No, that is, when the data is normal, arithmetic processing is performed on the data, the positive rate, etc. are determined, and the process branches to Sr1.

On the other hand, if the determination of Sr1 is YES, that is, if the data is abnormal, the process branches to 5TIO, stores the data in a list memory (not shown) attached to the MPU 21 (STIO), and stores the minute jJF fiTf area. It is determined whether or not to reset (STII).

When the judgment of this 5TII is Y F, S, 5T12
The process branches to Sr1 to reset the analysis area, performs recalculation based on the reset analysis area, and proceeds to Sr1.

When the determination of 5TII is No, the process branches directly to Sr1.

In Sr1, the calculation result is displayed on the CRT 23, and
This is printed out from the printer 24. IN<S
In r1, it is determined whether there is any sample remaining to be measured,
If this determination is YES, the process branches to Sr1, switches to the protocol corresponding to the next sample, and performs measurement. S
If the determination in r1 is NO, it is determined whether or not to end the measurement (Sr1). If this determination is YES, the measurement is ended, and if this determination is No, the process branches to Sr1. Sr1
Then, the sample is centrated and returned to the STI to start measurement again.

Although the above description is about continuous automatic measurement (automatic mode), for example, interrupt measurement is also possible. If you want to perform this interrupt measurement, remove the set sample, and there will be no sample, and Sr1
, Sr1, a sample for interrupt measurement is set at Sr1, and the mode is switched using STI to perform measurement.

Note that the parameters to be used are not limited to the four of forward scattered light intensity, 90° scattered light intensity, green fluorescence intensity, and red fluorescence intensity, and the design can be changed as appropriate.

(f) As described in detail of the invention, the cell analyzer of the present invention includes a sample supply means for sequentially aspirating a plurality of samples and sending the aspirated samples to the flow cell, and a cell analyzer supplied by the sample supply means. A measurement in which the cytooptical information processing means is set to the optimal measurement conditions according to the sample being used.

The present invention is characterized by comprising a condition setting means.

Therefore, the reliability of measurement results can be improved, and a large number of samples can be efficiently measured.

[Brief explanation of the drawing]

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 diagram explaining the configuration of the cell analyzer, and FIG. FIG. 2 is a schematic diagram of a cytogram showing an example of setting an analysis area. 2: Auto sampler 7: Sample pump, 10: Flow cell, 14: Laser, 15a15b15c1
5d: Photodetector: MPU 23; CRT. 24: Printer.

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 analyzing and 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. A cell analyzer comprising: an output means for outputting results; a sample supply means for sequentially aspirating a plurality of samples and sending the aspirated samples to the flow cell; and measurement condition setting means for setting optimum measurement conditions in the cell optical information processing means.