JP2625935B2 - Cell analyzer - Google Patents

Description

The present invention relates to a cell analyzer to which flow cytometry is applied, and more particularly, to optimal setting of measurement conditions.

(B) Conventional technology Flow cytometry is, for example, a method in which cells (or particles equivalent thereto) labeled with a fluorescent dye are caused to flow in a thin liquid stream, and cells that flow in a single row due to a hydrodynamic focusing effect. A cell is analyzed by irradiating each one with a beam light such as a laser, instantaneously measuring the intensity of scattered light or fluorescence generated from the cells, that is, cell light information. This flow cytometry has a feature that a large amount of cells can be analyzed at high speed and with high accuracy.

As a cell analyzer to which the above flow cytometry is applied, a flow cell for forming a thin liquid flow, a light source (for example, a laser) for irradiating a cell flowing in the flow cell with a light beam, and the light beam are irradiated. 2. Description of the Related Art An optical detector that detects cell light information from a cell and converts the light signal into an electric signal and a computer that performs analysis processing of the cell light information converted into the electric signal are known.

For example, in the case of analysis of a subset of lymphocytes in blood, a monoclonal antibody labeled with a fluorescent dye [in the case of double staining, for example, an OKT4 monoclonal antibody labeled with fluorescein isothiocyanate (FITC: green fluorescence), and Phycoerythrin (PE: red fluorescent) label OKT
8 monoclonal antibody] and a sample blood, and then subjected to hemolysis and used as a sample.

The sample cell flowing in the flow cell is irradiated with a laser beam, and four parameters, namely, forward scattered light intensity
I _0, 90 ° scattered light intensity I _90, the green fluorescence intensity I _g, red fluorescence intensity I _r is detected in the respective detector cell light information (hereinafter referred to simply as data) is obtained. Since this sample contains monocytes, granulocytes and the like in addition to lymphocytes, it is necessary to select data on lymphocytes from data on other cells.

Therefore, a cytogram was created with the forward scattered light intensity I ₀ and 90 ° scattered light intensity I ₉₀ as the orthogonal reference axis, and an analysis area called a window was set on this cytogram to select lymphocyte data. A known method is known (see, for example, JP-A-62-134559). Thus, the data of the sorted lymphocytes, green fluorescence intensity I _g, the analysis of the red fluorescence intensity I _r done, such as calculation of prevalence is performed.

(C) Problems to be Solved by the Invention In recent years, the above-mentioned cell analyzer automatically supplies a large number of samples in order to efficiently measure a large number of samples.
The introduction of a so-called autosampler is being considered. This autosampler is also preferable from the viewpoint of preventing biohazard since it can avoid direct contact between the sample and the operator.

However, there are many factors that affect the positive rate, and the measurement conditions differ depending on the sample processing method. For example, because the reactivity between various monoclonal antibodies and cells is different, measurement cannot be performed with the same detector with the same detection gain, and when detecting the fluorescence intensity due to the difference in the binding mode between the monoclonal antibody and the fluorescent substance. Correction calculation is required.

Therefore, it is necessary to select and set the optimal measurement conditions for each sample. However, this selection and setting has a lot of empirical knowledge, and conventionally, the operator performs the selection and setting work while monitoring. Was. However, this operation is skillful and laborious, and hinders improvement of the reliability of the measurement result and efficient measurement of a large number of samples. Therefore,
Even after introducing an autocypler and automating only the sample supply, the selection and setting of measurement conditions remains the same as before.
Improvements in measurement efficiency and reliability of measurement results cannot be expected.

The present invention has been made in view of the above, and an object of the present invention is to provide a cell analyzer capable of automatically setting optimum measurement conditions for each sample, improving the reliability of measurement results, and efficiently measuring a large number of samples. .

(D) Means and Action for Solving the Problems In order to solve the above problems, the cell analyzer of the present invention comprises a flow cell through which a cell (or equivalent) suspension liquid flows, and a cell flowing through the flow cell. A light source for irradiating a light beam, cell light information detecting means for detecting cell light information comprising one or more parameters for cells irradiated with the light beam, and cells detected by the cell light information detecting means Cell light information selecting means for selecting cell light information of a target cell population from light information, and cell light information detected by the cell light information detecting means and cell light information selected by the cell light information selecting means. A cell analyzer comprising: a cell light information processing means for performing an analysis process; and an output means for outputting a processing result of the cell light information processing means. Sample supply means for sending the aspirated sample to the flow cell; measurement condition setting means for setting the optimal measurement conditions according to the sample supplied by the sample supply means in the cell light information processing means; and the cell light information A parameter resetting unit for asking whether resetting of parameters relating to cell light information is necessary when an analysis processing result by the processing unit is abnormal.

In the cell analyzer of the present invention, the sample is sequentially supplied to the flow cell by the sample supply means, and the measurement is continuously performed. At this time, even if the processing method of the sample is different, the measurement corresponding thereto is performed. 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 the measurement result.

(E) Embodiment One embodiment of the present invention will be described below with reference to the drawings.

FIG. 2 is a diagram showing a configuration of a cell analyzer according to the embodiment. Reference numeral 2 denotes an autosampler, and its sample rack 2a
(Only one is shown) includes a plurality of sample containers 3,.
3 are loaded. The sample rack 2a is driven by a drive mechanism (not shown), and positions a designated sample immediately below the sample suction tube 4. Furthermore, this sample rack 2a
Is equipped with a shaking mechanism and a cooling mechanism (not shown), which shakes the sample periodically and keeps the sample in the loaded sample containers 3,... 3 at a low temperature (for example, 4 ° C. to 10 ° C.). I do.

The autosampler 2 includes, for example, five different processing methods α, for blood collected from one patient.
Samples obtained by processing with β, γ, δ, and ε are put into five different sample containers 3 and set. That is, five types of samples are set for one patient, and the total number of samples is obtained by multiplying the number of patients by five.

On the other hand, the sample suction tube 4 is connected to one port of the three-way valve 5. The other two ports of the three-way valve 5 can communicate the sample feeding tubes 6a and 6b with the sample suction tube 4, or the sample feeding tubes 6a and 6b. A sample pump 7 is provided at the other end of the sample feeding tube 6a. On the other hand, the other end of the sample feeding tube 6b is
It is open in the sheath liquid feeding tube 8.

One end of the sheath liquid feed tube 8 has a liquid feed pump 9
, And the other end is connected to the flow cell 10. The flow cell 10 is made of a material that transmits the wavelength of light to be used, such as quartz glass, and has an internal flow channel.
A sheath flow is formed in 10a, and cells or particles in the sample are transferred to the flow channel by the hydrodynamic focusing effect.
Flows in a line on the central axis of 10a.

The liquid flowing out of the flow cell 10 is guided to a waste liquid tube 11 and stored in a waste liquid tank 12. The cell analyzer has a sheath liquid tank (not shown), and the sample pump 7 and the liquid sending pump 9 are replenished with a sheath liquid.
Further, the liquid feeding system including the autosampler 2, the pumps 7, 9 and the like can be hermetically sealed, thereby preventing biohazard.

Around the flow cell 10, a laser (light source) 14 and photodetectors (cell optical information detecting means) 15a, 15b, 15c, 15d are arranged. The laser beam 1 from the laser 14 is applied to the cells (or particles) 1 flowing through the flow channel 10a.
Signal light is generated from the cell (or particle) 1,
Of these, the one in the forward direction is
The light is condensed on a and enters the photodetector 15a. Reference numeral 17 denotes a beam blocker for preventing the laser beam 1 from being incident on the linear photodetector 15a.

On the other hand, the signal light in the 90 ° direction from the cell 1 is transmitted through the lens 16b.
The light is focused. A part of this signal light is reflected by the dichroic mirror 18a, and the light detector 1 for detecting 90 ° scattered light is used.
It is incident on 5b. A part of the signal light transmitted through the dichroic mirror 18a is further reflected by another dichroic mirror 18b, transmitted through the filter 19a, and received by the photodetector 15c for green fluorescence. The signal light transmitted through the dichroic mirror 18b is transmitted through the filter 19b and received by the photodetector 15d for red fluorescence. Note that, for example, an argon laser or a helium neon laser is used for the laser 14, a photodiode is used for the photodetector 15a for forward scattered light,
A photomultiplier tube is applied to the other photodetectors 15b, 15c, and 15d.

The light receiving 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, forward scattered light intensity
A function of creating a cytogram or a histogram with respect to I ₀ , 90 ° scattered light intensity I ₉₀ and selecting data of a target cell (or particle) population. For the selected data, green fluorescence intensity I _g and red fluorescence intensity I _It has a function to determine the positive rate by analyzing _r , a function to control the autosampler 2, the sample pump 7, and the liquid sending pump 9, and the like.

A keyboard 22, a CRT 23, and a printer 24 are connected to the MPU 21. The keyboard 22 is used to specify the mode, select / set the protocol (measurement conditions), or send other commands to the MPU
It is for input to 1. The CRT 23 monitors the measurement, and the printer (output means) 24 prints out the processing result of the MPU 21, for example, a cytogram or a histogram.

Next, the operation of the cell analyzer of the embodiment will be described with reference to FIGS.

When the power of the cell analyzer is turned on, a ROM (not shown)
The program is read by 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. When the description is given on the assumption that the automatic mode has been selected, the automatic mode automatically and continuously measures the samples set in the autosampler 2 in the designated order. The sample specification is 5
A sample (5-α) treated with the treatment method α of the second patient,
The sample treated with the treatment method γ of the third patient (3-
γ), and so on. Of course, it is possible to specify that the same sample is measured twice.

In ST2, selection and setting of a protocol for the sample to be measured are performed. The protocol is a measurement condition such as the detection gain of the photodetectors 15a to 15d and the content of the correction operation.
The selection and setting of this protocol are determined in advance by the processing methods α to ε.
The optimum numerical value for the sample subjected to the test is input, and the protocol is automatically switched to the optimum protocol in the sample measurement order.
For example, when the sample is designated as (5-α), (3-γ),..., The protocol is sequentially switched to the protocol for the processing method α, the protocol for the processing method γ,.

In addition to the above, for example, when the measurement of the specified sample is completed, the protocol selection and setting may be performed in a protocol input waiting state, and the protocol may be input or the protocol may be input for each sample. You can do it too. Alternatively, the protocol can be selected and set by an external computer or the like.

The semi-automatic mode is a mode in which measurement is performed on only one designated sample. For example, if (4-β) is designated, among the fourth patient samples, those with the processing method β are aspirated. The protocol corresponding to the processing method β is automatically selected and the measurement is performed. On the other hand, in the manual mode, the measurement is performed only for one designated sample, but this is different from the semi-automatic mode in that the protocol is manually input.

When the processing of ST2 is completed, the sample rack 2a is driven,
First, the sample container 3 in which the sample to be measured is placed is located immediately below the sample suction tube 4. Then, the three-way valve 5 is switched so that the sample suction tube 4 and the sample 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 feeding tube 6a (ST3). .

Next, the three-way valve 5 is switched so as to communicate the sample sending tubes 6a and 6b, the sample pump 7 is driven to the sending side, and the sample is sent to the sending 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 10. In 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 a hydrodynamic focusing effect. Each of these cells is irradiated with a laser beam l,
Forward scattered light intensity I ₀ , 90 ° scattered light intensity I ₉₀ , green fluorescence intensity I
_g, red fluorescence intensity I _r is gradually being measured respectively (ST4).

The data measured in this way is processed according to a predetermined format. For example, in the analysis of leukocytes, a cytogram having a forward scattered light intensity I ₀ and a 90 ° scattered light intensity I ₉₀ is created (see FIG. 3). On this cytogram,
The distribution b of lymphocytes, the distribution c of monocytes, and the distribution d of granulocytes appear. Note that a is the distribution of red blood cell membrane components and the like called debris, and is often removed at the stage of noise processing. Of course, the format is not limited to the leukocyte analysis format.

In ST5, for example, the analysis areas B, C, and D are set for the lymphocyte distribution b, monocyte distribution c, and granulocyte distribution d, respectively, and the average intensity, standard deviation (SD), and coefficient of variation (CV) are set.
And the like are compared with a previously input reference value to determine whether the data is abnormal. When this determination is NO, that is, when the data is normal, the arithmetic processing of the data is performed to obtain a positive rate and the like, and the process branches to ST6.

On the other hand, if the determination in ST5 is YES, that is, if the data is abnormal, the process branches to ST10, where the data is stored in a list memory (not shown) attached to the MPU 21 (ST1).
0), it is determined whether or not to reset the analysis area (ST1)
1). If the determination in ST11 is YES, the process branches to ST12 to reset the analysis area, performs recalculation based on the reset analysis area, and proceeds to ST6. When the determination in ST11 is NO, the process branches to ST6.

In ST6, the calculation result is displayed on the CRT 23 and printed out from the printer 24. In the following ST7, it is determined whether or not the sample to be measured remains, and this determination is made by YE
In the case of S, the process branches to ST2, and the measurement is performed by switching to the protocol corresponding to the next sample. If the determination in ST7 is NO, it is determined whether to terminate the determination (ST8). If this determination is YES, the determination is terminated, and if NO, ST9
Branch to In ST9, the sample is set, the process returns to ST1, and the sample is started again.

In the above description, automatic setting is performed continuously (automatic mode). However, 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.
7. Via ST8, the sample for interrupt measurement is set in ST9, and the mode is switched in ST1 to perform measurement.

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

(F) Effects of the Invention As described above, the cell analyzer of the present invention suctions a plurality of samples sequentially and feeds the sucked samples to the flow cell, and the sample analyzer supplies the sample to the flow cell. Measurement condition setting means for setting optimal measurement conditions according to the sample to the cell light information processing means, and necessity of resetting parameters related to cell light information when the analysis processing result by the cell light information processing means is abnormal. And a parameter resetting means for asking the user. Therefore, the reliability of the measurement result can be improved, and a large number of samples can be measured efficiently. In addition, when the measurement result is determined to be abnormal, the parameter can be reset freely, which also contributes to efficient measurement.

[Brief description of the drawings]

FIG. 1 is a flowchart illustrating the operation of the cell analyzer according to one embodiment of the present invention, FIG. 2 is a diagram illustrating the configuration of the cell analyzer, and FIG. It is a schematic diagram of the cytogram which shows an example of a distribution area setting. 2: Autosampler, 7: sample pump, 10: flow cell, 14: laser, 15a / 15b / 15c / 15d: photodetector, 21: MPU, 23: CRT, 24: printer.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Mitsuo Nishimura, 3rd Hanazono Nakamimoncho, Ukyo-ku, Kyoto, Kyoto Prefecture Inside Tateishi Life Science Research Institute, Inc. No. 3, Tateishi Life Science Research Institute, Inc. (72) Inventor Fumio Onuma, No. 3, Hanazono Nakamimoncho, Ukyo-ku, Kyoto, Kyoto Prefecture Within Tateishi Life Science Research Institute, Inc. (72) Inventor Shinichi Hirako, Ukyo, Kyoto, Kyoto 3 in Tateishi Life Science Research Institute, Tateishi Life Science Institute, Inc. (72) Inventor Kunio Kaede, 3 in Tateishi Life Science Research Institute, Hanazono, Kyoto, Kyoto, Kyoto Prefecture 62-134559 (JP, A) JP-A-63-167273 (JP, A)

Claims (1)

(57) [Claims] 1. A flow cell through which a cell suspension flows, a light source for irradiating a cell flowing in the flow cell with a light beam, and cell light information comprising one or more parameters for the cell irradiated with the light beam. Cell light information detecting means for detecting the cell light information detected by the cell light information detecting means,
A cell light information selecting means for selecting cell light information of a target cell population; and a cell for analyzing and processing the cell light information detected by the cell light information detecting means and the cell light information selected by the cell light information selecting means. In a cell analyzer, comprising: an optical information processing unit; and an output unit for outputting a processing result of the cell optical information processing unit, wherein a plurality of samples are sequentially sucked, and a sample is sent to the flow cell. Supply means; measurement condition setting means for setting optimal measurement conditions according to the sample supplied by the sample supply means in the cell light information processing means; and an analysis result by the cell light information processing means is abnormal. And a parameter resetting unit for inquiring whether resetting of parameters relating to cell light information is necessary.