JP3092232B2 - Cell analyzer - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cell analyzer to which flow cytometry is applied, and more particularly, to a method of correcting the measured cell number to a reference cell number in the process of processing the light information of the cells and unifying them. The present invention relates to a cell analyzer that performs

[0002]

2. Description of the Related Art Flow cytometry is a method in which a sample containing cells (or particles equivalent thereto) labeled with, for example, a fluorescent dye is caused to flow in a thin liquid stream, and the cells flow in a single row by a hydrodynamic focusing effect. Is irradiated with a beam light such as a laser, and the intensity of scattered light or fluorescence generated from the cells, that is, cell light information is instantaneously measured to analyze the cells. This flow cytometry has a feature that a large amount of cells can be analyzed at high speed and with high accuracy.

A cell analyzer to which the above-mentioned flow cytometry is applied, when a sample contains a plurality of cell populations, analyzes one or more cell populations based on cell light information. And a device having a so-called auto-trigger function for automatically setting cell light information belonging to the analysis region has been filed by the present applicant (Japanese Patent Application Nos. 62-22884 and 63-193).
033, Japanese Patent Application No. 63-193072).

[0004] In order to maintain the objectivity and compatibility of the analysis, it is desirable that the number of cells in the analysis area is uniform for each sample. An apparatus having a unified function has also been filed by the present applicant (Japanese Patent Application No. 63-3209).
No. 15). Furthermore, since it is difficult to set an analysis area when an abnormal sample is measured as a sample, an apparatus having a function of using an analysis area set in advance has also been filed by the present applicant (Japanese Patent Application No. Hei. 149162).
In this cell analyzer, the analysis area of the normal sample measured immediately before is stored, and the number of cells is measured in the stored analysis area. If the sample measured immediately before is an abnormal sample, the number of cells is measured using an analysis area that has been manually set in advance. The counting of the number of cells n ′ in this analysis region is performed up to the specified number of cells n _a0 with a margin of α (n _a0 + α) in both the normal sample and the abnormal sample. ' _Can be adjusted to _na0 (see FIG. 4).

[0005]

By the way, a sample is often prepared from human blood. The blood reflects variously the human pathological condition, and the scattered light cytogram of fluorescence cytogram and the cell of fluorescence cytogram are used. This is because changes often appear in the group pattern. There is some information on various blood cells contained in human blood, but in lymphocyte subset tests, information is often compared at a positive rate, making it difficult to directly compare with existing information or understanding information from multiple angles It may not be possible. In addition, sometimes compared with the measured cell number as a reference, the number of cells to be compared is different, if the reliability of the numerical value itself of the number of cells to be compared is insufficient, the objectivity of information, Compatibility and reliability may be suspected. Further, when the measured cell number does not reach the unified cell number, such a situation may be present in a sample of a patient whose disease state is being monitored, for example, a patient who has been administered an anticancer drug for leukemia, but the reliability of the analysis indicates Then, there is a problem that reliability is reduced. In addition, if the test sample is normal but diluted too much during sample preparation, or if the sample aspiration operation could not be completed by mistake, the apparent number of cells will be measured. In addition, there is a problem that the sample must be re-prepared or re-measured, and the efficiency of automation cannot be improved.

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a cell analyzer capable of sufficiently ensuring the reliability and compatibility of analysis information even when the number of target cells in a measurement sample does not reach the unified cell number. Is to provide.

[0007]

Means for Solving the Problems In order to solve the above problems, the cell analyzer of the present invention has the following configuration (7) in addition to the conventional configuration [the following configurations (1) to (6)]. , (8)
It is characterized by having. That is, (1) a flow cell through which a control sample such as a cell suspension to be measured flows, (2) a light source that irradiates a cell or the like flowing through the flow cell with a light beam, and (3) a light source that irradiates the light beam. Cell light information detecting means for detecting cell light information comprising one or more parameters for each cell or the like; and (4) cell light of a target cell population from cell light information detected by the cell light information detecting means. Cell light information selecting means for selecting information; and (5) cell light information processing means for arithmetically 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. And (6) an output means for outputting the processing result of the cell light information processing means. (7) The number of target cells (N) in a control sample having a known concentration. ₀ ) Constant, and the number of cells and the analyte concentration storing means for storing (N _0), (8) the number of cells the target cells in the control sample measurement conditions identical test sample using the measurement conditions and (n _{a0) Is} measured, and the number of cells (n _a0 ) is multiplied by N ₀ / n _a0 to unify the number of cells of the test sample (n _a0 ) to the number of cells of the control sample (N ₀ ) by calculation. Means.

[0008]

In the cell analyzer of the present invention, the cell number (N ₀ ) of the target cells in the control sample is measured and stored by the sample concentration storage means, and the measurement conditions of the control sample are determined by the cell number unifying means. Under the same measurement conditions, the number of target cells in the test sample (n _a0 ) was measured, and the number of cells of the test sample (n _a0 )
By multiplying by ₀ / n _a0 , the number of cells of the test sample (n _a0 ) can be matched with the number of cells of the control sample (N ₀ ) by a correction operation. For this reason, even if the number of cells in the test sample is less than the reference control sample, especially when the test sample is normal and the cell count is low for some reason, or in patients undergoing leukemia treatment, In the case of the sample, the number of cells of the test sample is always unified with the number of cells of the control sample by calculation, and the number of cells of the test sample is unified with the number of cells obtained from the control sample of known concentration. Automation and efficiency are not hindered, and data compatibility, objectivity, reliability, etc. are maintained.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, a cell analyzer according to the present invention will be described based on embodiments. FIG. 1 is a schematic diagram showing a configuration of a cell analyzer as one embodiment. The sample cells 1 are supplied by an autosampler 2, and a plurality of sample containers 3,..., 3 are loaded in a sample rack 2a of the autosampler 2. The sample rack 2a is driven by a driving mechanism (not shown), and positions a designated sample immediately below the sample suction tube 4. Further, the autosampler 2 is provided with a shaking mechanism and a cooling mechanism (neither is shown). The autosampler 2 shakes the sample periodically and loads the loaded sample containers 3,.
The sample in 3 is kept at a low temperature (for example, 4 to 10 ° C.).

The sample suction tube 4 is connected to one of the three-way valves 5.
Connected to two ports. The sample sending tubes 6a and 6b are connected to the other two ports of the three-way valve 5, respectively. The sample sending tube 6a and the sample suction tube 4 or the sample sending tubes 6a and 6b communicate with each other. Can be. A sample pump 7 is provided at the other end of the sample feeding tube 6a. On the other hand, the sample feeding tube 6
The other end of b opens into the sheath liquid feeding tube 8.

The sheath liquid feed tube 8 has one end connected to the liquid feed pump 9 and the other end connected to the 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 cells (or particles) 1 in a sample move on the central axis of the flow channel 10a by a hydrodynamic focusing effect. Shed in a line.

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 includes a sheath liquid tank (not shown), and the sample pump 7 and the liquid sending pump 9 are replenished with a sheath liquid. Also, these pumps 7, 9 are advantageously syringe pumps. Further, the liquid sending system including the autosampler 2, the pumps 7, 9 and the like can be sealed, thereby preventing biohazard.

Around the flow cell 10, a laser (light source) 14, a photodetector (cell light information detecting means) 15a,
5b, 15c and 15d are provided. The laser beam L from the laser 14 is applied to the cells (or particles) 1 flowing through the flow channel 10a. This cell (or particle) 1
Rather, a signal light is generated, and the forward one of them is collected as forward scattered light by the lens 16a.
The light enters the photodetector 15a. Note that a beam blocker 17 for preventing the laser beam L from directly entering the photodetector 15a is provided.

On the other hand, the signal light in the 90 ° direction from the cell 1 is collected by the lens 16b. A part of this signal light is reflected by the dichroic mirror 18a and is incident on the photodetector 15b for detecting 90 ° scattered light. Part of the signal light transmitted through the dichroic mirror 18a is further reflected by another dichroic mirror 18b, passes through the filter 19a, and is received by the photodetector 15c for green fluorescence. The light transmitted through the dichroic mirror 18b is transmitted through the filter 19b and received by the red fluorescence photodetector 15d. For example, an argon laser or a helium neon laser is used as the laser 14, a photodiode is used as the photodetector 15a for forward scattered light, and a photomultiplier is used as the other detectors 15b, 15c, and 15d.

Photodetectors 15a, 15b, 15c, 15d
Is amplified by the signal processing circuit section 20 to remove noise, and then converted into an analog / digital (A / D) converter 2
The signal is converted into a digital signal by 1 and is taken into the MPU 22. The MPU 22 has various functions such as a function related to an auto trigger, a function of storing the number of cells, and a function of correcting and calculating the number of cells. Other functions include a function of performing fluorescence analysis on optical information of a target cell population, a function of controlling the sample pump 7, the liquid sending pump 9, and the autosampler 2.

The MPU 22 includes a floppy disk drive 23, a keyboard 24, a CRT 25, and a printer 26.
Is connected. The floppy disk drive 23
This is for storing measurement conditions (protocol), measurement data, and the like on a floppy disk. Keyboard 24
Can be used to select / set protocol or other commands
This is for inputting to U22. The CRT 25 is for monitoring the measurement, and the printer (output means) 26 is for printing out a processing result of the MPU 22, for example, a cytogram or a histogram.

Next, the operation of the cell analyzer of the embodiment will be described with reference to FIG.
And a flowchart shown in FIG. First, a sample sample that has been subjected to processing according to the purpose of analysis is placed in each sample container 3, and the sample rack 2a
To be loaded. For example, a sample for analysis of a lymphocyte subset is prepared by reacting a blood sample of a patient with an OKT4 monoclonal antibody labeled with fluorescein isothiocyanate (FITC) and an OKT8 monoclonal antibody labeled with phycoerythrin (PE). , Obtained by hemolysis.

When the power of the cell analyzer is turned on, RO
A program is read into MPU 22 from M (not shown), and the system starts up [Step (hereinafter referred to as ST)].
1, see FIG. 2). Here, if the measurement target is a control sample,
That is, when a sample whose concentration is known is measured by this apparatus using a blood cell counter or the like (ST2), the protocol is selected.
It is set (ST3). This protocol uses photodetector 1
The content includes measurement conditions such as detection gains and correction calculations for 5a, 15b, 15c, and 15d. This is because the sample processing method differs depending on the measurement purpose. For example, since the reactivity between the monoclonal antibody used in each treatment and cells and the like is different, the photodetectors 15a, 15b, 15
It is necessary to change the detection gains of c and 15d, and the binding mode between each monoclonal antibody and the fluorescent dye is different, so that the correction calculation also needs to be changed accordingly.

In the next ST4, an analysis area is manually set for measurement. The analysis area setting function is used, for example, for measurement of blood cell control, and performs counting and calculation processing of blood cell control. The blood cell control is measured as a control sample (ST5), and after the measurement is completed, the calculation in the analysis area is performed (ST6). ST
In 7, the calculation result is stored as needed. The stored contents are stored in the MPU 22 and read out when the unified calculation of the number of cells is performed, and used for the calculation.

In step ST2, for example, if the control sample has already been measured and the information on the control sample has been stored and the control sample measurement is not to be performed, the protocol is selected and set in step ST8 to perform the measurement of the test sample. Proceed (ST10). After the measurement of the control sample, the process proceeds to ST9 to select whether or not to use the same protocol. If NO, the process proceeds to ST8, and if YES, the process proceeds to ST10. Then S
At T10, select whether or not to perform an auto trigger, and select NO.
If it is, go to ST13, and if YES, go to ST11.
In ST11, it is determined whether or not the measurement is a new measurement. If the measurement has been performed before, the analysis area input in the previous measurement in ST12 is read and set. In the case of a new measurement, the analysis area is manually set (ST1).
3). Thereafter, measurement is started (ST14), and ST15
Select whether to perform cell number unification with. When performing this, the auto-trigger calculation and the unified cell count calculation are performed (ST1).
6). If the cell number unification is not performed, only the auto trigger calculation is performed (ST19).

The auto-trigger calculation is performed by a method disclosed in, for example, Japanese Patent Application Nos. 63-172096 and 63-193072. FIG. 4 is a schematic diagram showing the work of unifying the number of cells, and FIG. 5 is a diagram showing forward scattered light intensity I ₀ and 90 ° scattered light intensity I ₉₀ , which are assumed to represent the size and internal structure of the cell. Is a cytogram using as orthogonal coordinate axes. However, in FIG. 5, b shows the distribution of lymphocytes, c shows the distribution of monocytes, and d shows the distribution of granulocytes. First, on this cytogram, an analysis area B ₀ including the distribution b of lymphocytes is set, and measurement is continued until the number of cells belonging to this analysis area B ₀ becomes n ′. This cell number n ′ is a number sufficiently larger than the final cell number n _a0 , and the measurement is continued until n′≫n _a0 + α, and information is collected.

On the other hand, in the unified cell number calculation, in order to set the cell number n 'to the cell number n _a0 of the cells in the final fraction B ₀ ', the cells shown in FIGS. 4 (a), (b) and (c) are used. The portion exceeding the cell number n _a0 with respect to the cell number n ′ is discarded by any method.
If the cell number n ′ is less than the cell number n _a0 even after performing the above measurement (n ′ <n _a0 ), the cell number n stored in ST7
_a0 is read out, and the cell number n 'is multiplied by a coefficient of _na0 / n', and is unified to the cell number _na0 . Therefore, as a result of analyzing a specimen using a plurality of monoclonal antibodies in the lymphocyte subset test, the number of cells n
_{1 ', n 2', ···} , ' if are obtained, these cell numbers _{n a0} / n' n n is multiplied by the coefficient, n ₁ '·
(N _a0 / n '), n ₂ ' · (n _a0 / n '), ..., n
_n ′ · (n _a0 / n ′). As a result, in each case, the number n ′ of cells in the test sample is unified to the number n _a0 of cells in the control sample as a reference.

In each case, the fluorescent characteristic analysis is performed on the information processed in ST16 or ST19 (ST17, ST20). In this analysis, for example, respective histograms are created for the green fluorescence intensity and the red fluorescence intensity, and the positive rate is calculated based on the histograms. This result is displayed on the CRT 25 in the unified cell number mode (S
T18), displayed in the normal mode (ST21), and both are printed out by the printer 26 (ST2).
2). After that, MPU2 determines whether there is more measurement sample.
2 (ST23), and if there is a sample, the process returns to ST9. If there is no sample, the analysis is terminated (ST24).

In the above-described embodiment, the case where the cell count is unified is lymphocytes. However, the measurement can be similarly performed for monocytes, granulocytes, and leukocytes.

[0025]

As described above, the cell analyzer of the present invention is provided with the sample concentration storage means and the cell number unifying means, so that the cell number of the test sample matches the cell number of the control sample having a known concentration. Therefore, even if the number of cells of the test sample does not reach the reference control sample, the number of cells of the test sample can be unified with the number of cells of the control sample by the correction operation. Therefore, since the information comparison and information calculation are performed after the number of cells in the cell population to be analyzed is always fixed, the objectivity and compatibility of the cell light information are maintained, and the cell light information with higher accuracy is obtained. Is obtained.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a configuration of a cell analyzer according to one embodiment of the present invention.

FIG. 2 is a flowchart illustrating the overall operation of the cell analyzer according to the present invention.

FIG. 3 is a flowchart following the flowchart shown in FIG. 2;

FIG. 4 is a schematic diagram for explaining the work of unifying the number of cells.

FIG. 5 is a two-dimensional distribution diagram of cell groups displayed on cytograms I ₀ -I ₉₀ and cytograms.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Sample cell 10 Flow cell 14 Laser 15a, 15b, 15c, 15d Photodetector 20 Signal processing circuit part 22 MPU 25 CRT 26 Printer

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 15/14

Claims (5)

(57) [Claims] 1. A flow cell through which a control sample such as a cell suspension to be measured flows, a light source irradiating a cell or the like flowing through the flow cell with a light beam, and each cell irradiated with the light beam. Cell light information detecting means for detecting cell light information comprising one or more parameters, and cell light information selecting means for selecting cell light information of a target cell population from the cell light information detected by the cell light information detecting means Cell light information processing means for arithmetically processing the cell light information detected by the cell light information detection means and the cell light information selected by the cell light information selection means; processing results in the cell light information processing means in the cell analyzer comprising and an output means for outputting a concentration measure the cell number (N ₀₎ of the target cells in the control sample is known, stores this cell number (N ₀₎ search And body density storage unit, using the measurement conditions identical measurement conditions and the control sample to determine cell numbers of target cells in a test sample _{(n a0), N 0 /} n in the cell number (n _a0) A cell analyzer comprising: a cell number unifying unit that multiplies _a0 by calculation to make the number of cells of the test sample (n _a0 ) equal to the number of cells of the control sample (N ₀ ) by calculation. 2. The cell analyzer according to claim 1, wherein the target cells selected by said cell number unifying means are lymphocytes. 3. The cell analyzer according to claim 1, wherein the target cells selected by said cell number unifying means are monocytes. 4. The cell analyzer according to claim 1, wherein the target cells selected by said cell number unifying means are granulocytes. 5. The cell analyzer according to claim 1, wherein the target cells selected by said cell number unifying means are leukocytes.