JPH0769325B2 - Cell analyzer - Google Patents

Description

TECHNICAL FIELD The present invention relates to a cell analyzer to which flow cytometry is applied, and more specifically, a process for selectively collecting cell light information about a specific cell population in a sample. Regarding

(B) Conventional technology In flow cytometry, for example, cells labeled with a fluorescent dye (or particles equivalent thereto) are caused to flow in a thin liquid flow,
By irradiating laser light to each cell flowing in a row by the hydrodynamic focusing effect, the intensity of scattered light or fluorescence generated from the cell, that is, cell light information is instantaneously measured.
It analyzes cells. This flow cytometry has the feature that it can analyze a large number of cells at high speed and with high accuracy.

As a cell analysis device to which the above flow cytometry is applied, a flow cell for forming a fine liquid flow, a light source (for example, a laser) for irradiating a light beam to cells flowing in the flow cell, and this light beam are irradiated. 2. Description of the Related Art There is known a device including a photodetector that detects cell light information from cells and converts the cell light information into an electric signal, and a computer that performs analysis processing of the cell light information converted into the electric signal.

In this conventional cell analyzer, a cell suspension (sample) dyed with a fluorescent dye is made to flow in a flow cell together with a sheath liquid. A sheath flow is formed in the flow cell, and cells flow in a line on the central axis of the flow cell due to the hydrodynamic focusing effect.

The intensity of scattered light and fluorescence generated by irradiating these cells with a light beam is detected by a photodetector such as a photomultiplier tube as a parameter constituting cell light information.

Now, when a plurality of cell populations are contained in the cell suspension, it may be desired to analyze one of these cell populations. For example, in the case of analysis of lymphocyte subsets in human blood, hemolyzed blood is used as a sample, and this sample contains monocytes and granulocytes in addition to lymphocytes. Therefore, it is necessary to select lymphocytes from the cell light information obtained by the measurement.

Therefore, a so-called window method is known as a method of selecting and collecting cell light information of a necessary cell population (see, for example, JP-A-62-134559). In this window method, an operator sets an analysis area called a window in a space having one or more parameters of cell light information as a coordinate system, and the light information of cells belonging to this area is used as a target cell population. Cell light information was collected.

For example, in the case of analysis of a subset of the above lymphocytes,
Of the cell light information, two parameters of 90 ° scattered light intensity I ₉₀ and forward scattered light intensity I ₀ are used. FIG. 6 shows a schematic diagram of a cytogram with I ₉₀ on the horizontal axis and I ₀ on the vertical axis. b shows the distribution of lymphocytes, c shows the distribution of monocytes, and d shows the distribution of granulocytes. It should be noted that a represents the distribution of debris, but this debris is composed of the membrane components of red blood cells and the like, and is usually removed in the stage of noise processing.

To analyze lymphocytes, a control sample (a sample of a healthy person) is used to set a window e as shown by a chain double-dashed line in FIG. Cell light information belonging to this window e is collected from the measured whole cell light information, analysis processing is performed for fluorescence characteristics, and the results are displayed on the CRT.
Output from the printer.

However, in the window method, it may be impossible to collect the cell light information of the target cell population unless the operator changes the window depending on the sample. For example, in the analysis of lymphocyte subsets, the position, size, and shape of the distribution b of lymphocytes shown in FIG. 6 change depending on the sample, and the operator has to change the window e accordingly. Such window changes prevent efficient and automatic measurement of many samples.

Therefore, the applicant of the present application has already applied for a cell analyzer that can automatically collect cell light information without the operator setting a window (Japanese Patent Application No. 62-22884).
issue). This cell analysis device uses 1 or 2 of the cell light information.
Histograms are created for the above parameters, the minimum points of these histograms are extracted, the cell population is fractionated based on these minimum points, and one or more fractions are set as an analysis region, and the optical information of the cells belonging to this is extracted. Are collected as cell light information of the target cell population.

For example, in the case of the analysis of the above lymphocyte subset, 90
A histogram of the scattered light intensity I ₉₀ and the forward scattered light intensity I ₀ is created [see FIGS. 7 (a) and 7 (b)]. Then, the minimum points p ₁ , p ₂ , p ₃ , and the minimum points P ₄ and P ₅ are extracted from the histogram of I ₀ from the histogram of I ₉₀ . If these minimal points p _{1 to} p ₅ are represented on the I ₉₀ and I ₀ cytograms, they are fractionated into the fractions shown by the broken line in FIG. Since the lymphocyte distribution b is included in the fraction B, the light information of cells belonging to this fraction B, that is, I ₉₀ is p ₁ or more and p ₂ or less, and I ₀ is p ₄ or more p
The light information of cells that is ₅ or less is retrieved from the measured light information of all cells, and the light information of lymphocytes is collected.

(C) Problem to be Solved by the Invention In the above-described conventional cell analyzer, the cell light information collecting means collects cell light information of the target cell population based on the fractions. This fraction is shown in FIG. As shown, it is a rectangular area on the cytogram. Therefore, this fraction does not perfectly match the shape of the distribution of the target cell population, and the optical information of unnecessary cells is included in the collected optical information of the target cell population, and the accuracy of analysis decreases. There was a problem.

A contour tracking method is known as a method for obtaining an analysis region more suited to the shape of the distribution of the target cell population, but it requires time for calculation processing and is not preferable in terms of efficiency of analysis.

The present invention has been made in view of the above, and an object of the present invention is to provide a cell analyzer that can determine a fraction more suitable for the distribution of a target cell population in a shorter processing time.

(D) Means for Solving the Problems In order to solve the above problems, the cell analysis device of the present invention aims at observing cell light information obtained from each cell by irradiating a cell suspension flowing in a flow cell with a light beam. In the cell analyzer that collects the cell light information of the cell population to be collected by the cell light information collecting means, and performs the analysis processing, the cell light information collected by the cell light information collecting means, in the fraction of the cell population. Highest degree numerical point extraction means for extracting the highest degree numerical point on the relevant cytogram, and a straight line forming a straight line on the cytogram from the highest degree numerical point extracted by the highest degree numerical point extraction means Forming means, boundary point extracting means for extracting boundary points from the histograms on the straight lines formed by the straight line forming means, and boundary points extracted by the boundary point extracting means A final fraction an area formed by connecting, is characterized in that a second cell light information collecting means for collecting cell light information of interest cell population based on the final fraction.

(E) Action The action of the cell analyzer of the present invention will be described with reference to FIG. 1 and FIG. 2 corresponding to the embodiment, with respect to the fraction B relating to cell light information obtained from cells by irradiation of a light beam. After the cell light information collecting means collects the cell light information of the target cell population, the highest numerical point q is extracted on the cytogram (see FIG. 1), and from this point q, the straight lines l ₁ to l are radiated. ₁₂
Is formed. The histograms on the straight lines l _{1 to} l ₁₂ are, for example, as shown in FIGS. 2 (a), (b) and (c). When a point at which this histogram intersects with a predetermined threshold value or a minimum point X of the histogram is extracted as a boundary point r and the boundary points r _{1 to} r ₁₂ for the respective straight lines l _{1 to} l ₁₂ are connected,
A fraction B'more suited to the distribution of the target cell population is obtained. If light information of the target cell population is further collected by the second cell light information collecting means based on this fraction, the light information of unnecessary cells and the like is not included in the collected cell light information, and the analysis accuracy is improved. Can be expected.

That is, in the cell analyzer described in the above-mentioned Japanese Patent Application No. 62-22884, the optical information (noise) of unwanted cells existing outside the area of the objective cell population within the rectangular fraction is also the optical information of the objective cell population. However, in the present invention, since the cell light information of the target cell population is collected by the second cell light information collecting means based on the final fraction formed by connecting the boundary points, the final fraction is initially The region becomes narrower than the rectangular fraction of, and is closer to the region of the target cell population, and noise contained outside the region of the target cell population in the fraction is not extracted by the second cell light information collecting means, Only the necessary information is extracted, the noise removal capability is superior to that of the above-described conventional cell analyzer, and the analysis accuracy is improved.

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

In this embodiment, the cell analysis device uses forward scattered light intensity I ₀ , 90 ° as cell light information (hereinafter sometimes simply referred to as data).
The scattered light intensity I ₉₀ , the fluorescence intensity I _{r of} two different colors, and the total of four parameters of I _g are adopted.

FIG. 3 is a diagram showing the configuration of the cell analyzer of the example.
Reference numeral 2 is an auto sampler, and the sample rack 2a is loaded with a plurality of sample containers 3 ,. The sample rack 2a is driven by a drive mechanism (not shown) to position a designated sample directly below the sample suction tube 4. Further, the auto sampler 2 is provided with a shaking mechanism and a cooling mechanism (not shown), and shakes the sample at regular intervals, and the samples in the loaded sample containers 3, ... Hold at ~ 10 ° C).

The sample suction tube 4 is connected to one port of the three-way valve 5. Sample feeding tubes 6a and 6b are respectively connected to the other two ports of the three-way valve 5, and the sample feeding tube 6a and the sample suction tube 4 or the sample feeding tubes 6a and 6b are connected to each other. You can 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 is opened inside the sheath liquid feeding tube 8.

The sheath liquid feeding tube 8 has one end connected to the liquid feeding pump 9 and the other end connected to the flow cell 10. The flow cell 10 is made of quartz glass or the like, a sheath flow is formed in the flow channel 10a inside, and cells or particles in the sample flow in a line on the central axis of the flower 10a by the hydrodynamic focusing effect. Be done.

The liquid flowing out from the flow cell 10 is guided to the waste liquid tube 11 and stored in the waste liquid tank 12. The cell analyzer is equipped with a sheath liquid tank (not shown), and the sample pump 7 and the liquid feed pump 9 are replenished with the sheath liquid.
Further, the liquid feeding system including the auto sampler 2, the pumps 7, 9 and the like can be hermetically sealed, and biohazard can be prevented.

A laser (light source) 14 and photodetectors (cell light information detecting means) 15a, 15b, 15c, 15d are arranged around the flow cell 10. The laser beam l from the laser 14 is a flow channel
The cells (or particles) 1 flowing through 10a are irradiated. Signal light is generated from the cells (or particles) 1, of which the light in the forward direction is focused on the lens 16a as forward scattered light and is incident on the photodetector 15a. 17
Is a beam blocker that prevents the laser beam 1 from directly entering the photodetector 15a.

On the other hand, the signal light in the 90 ° direction from the cell 1 is condensed by the lens 16b. A part of this signal light is reflected by the dichroic mirror 18a, and a photodetector 15b for detecting 90 ° scattered light is provided.
Incident on. A part of the signal light transmitted through the dichroic mirror 18a is 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 passes through the filter 19b and is received by the photodetector 15d for red fluorescence. Incidentally, for example, the laser 14 is an argon laser or a helium neon laser,
A photodiode is applied to the photodetector 15a for the forward scattered light, and a photomultiplier tube is applied to the other detectors 15b, 15c, and 15d.

The received light signals of the photodetectors 15a, 15b, 15c, 15d are converted into digital signals by an analog / digital converter (not shown) and taken into the MPU 21. MPU21 is the forward scattered light intensity
I ₀ , 90 ° scattered light intensity I ₉₀ A function to create cytograms and histograms for each, a function to extract local minimum points from this histogram and fractionate cell light information into each fraction, belonging to this fraction A function of collecting cell light information, a function of determining a final fraction of the collected cell light information, a function of collecting cell light information belonging to this final fraction, and analyzing fluorescence characteristics, the sample pump 7. It has a function of controlling the liquid sending pump 9 and the auto sampler 2.

A keyboard 22, a CRT 23, and a printer 24 are connected to the MPU 21. The keyboard 22 is for inputting selection / setting of protocols (measurement conditions) or other commands to the MPU 21. The CRT 23 is for monitoring the measurement, and the printer (output means) 24 is for printing out the processing result of the MPU 21, for example, a cytogram or a histogram.

Next, the operation of the embodiment cell analyzer will be described.

First, the samples that have been processed according to the purpose of analysis are placed in the sample containers 3 and loaded into the sample rack 2a. For example, a sample for analysis of lymphocyte subsets
Fluorescein isothiocyanate (FIT
It is obtained by reacting the OKT4 monoclonal antibody labeled with C) with the OKT8 monoclonal antibody labeled with phycoerythrin (PE) and then subjecting to hemolysis.

When the power of the cell analyzer is turned on, the program is read from the ROM (not shown) into the MPU 21, and the system starts up (step (hereinafter referred to as ST) 1, see FIG. 4 (a)).
Next, a protocol suitable for the sample to be measured is selected and set. This protocol includes photodetectors 15a, 15b, 15
It includes the measurement conditions such as the detection gain of c and 15d and correction calculation. This is because the treatment method of the sample differs depending on the measurement purpose, for example, because the reactivity of the monoclonal antibody used for each treatment with cells is different,
It is necessary to change the detection gain of the photodetectors 15a, 15b, 15c, and 15d, and since the binding modes of each monoclonal antibody and the fluorescent dye are different, the correction calculation also needs to be changed accordingly.

When the process of ST2 is completed, the sample rack 2a is driven, and the sample container 3 in which the sample to be measured first is placed is positioned directly below the sample suction tube 4. Then, the three-way valve 5 is switched so that the sample suction tube 4 and the sample solution feeding tube 6a communicate with each other, the sample suction tube 4 descends into the sample container 3, and the sample pump 7 is driven to the suction side to Is sucked into the sample delivery tube 6a (ST3).

Next, the three-way valve 5 is switched so as to connect the sample delivery tubes 6a and 6b, and the sample pump 7 is driven to the delivery side,
The sample is sent to the liquid sending tube 8. On the other hand, the liquid delivery pump 9
Is driven to the liquid sending side, and the sheath liquid is sent to the flow cell 10. A sheath flow is formed in 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. The laser beam 1 is irradiated to each of the cells, and the forward scattered intensity I ₀ , the 90 ° scattered light intensity I ₉₀ , the green fluorescent intensity I _g , and the red fluorescent intensity I _r are measured (ST4).

In ST5, MPU21 is forward scattered light intensity I ₀ , 90 ° scattered light intensity
A cytogram with I ₉₀ as the orthogonal axis (see FIG. 6) and respective histograms (see FIGS. 7 (a) and (b)) are created. Then, in ST6, it is determined whether the minimum point can be extracted from the I ₉₀ histogram.

When the sample is normal, three minimal points p ₁ , p ₂ , and p ₃ can be extracted from the I ₉₀ histogram [in the case of lymphocyte subset analysis, see FIG. 7 (a)]. In this case, ST9
If the sample is abnormal and the local minimum point cannot be extracted, the process branches to ST7 to notify the operator of the abnormality.
This notification is performed by displaying on the CRT 23 or by voice. Then, the operator manually corrects the fraction (ST8), and proceeds to ST9.

In ST9, the minimum points p ₁ , p ₂ , and p ₃ are extracted from the I ₉₀ histogram, and the data with I ₉₀ between p ₁ and p ₂ are selected and collected from all the measurement data of the sample. Fig. 5 (a)
(B) shows a histogram of I ₉₀ and I ₀ for this sorted and collected data. Furthermore, the minimum point is extracted from the histogram of I ₀ shown in FIG.
The fraction B is finally determined based on these minimum points (ST10, see also FIG. 6).

In subsequent ST11, the calculation for the final fraction is performed on the data in the fraction determined in ST10. This calculation will be described with reference to FIG. 4 (b). First, the highest numerical value point q is extracted from the data in the fraction determined in ST10 (ST111). Then, from this point q, straight lines 1 are radially formed at constant angles θ (ST112). For example, in the case of lymphocyte subset analysis, as shown in FIG.
The highest numerical value point q is extracted in the fraction B, and straight lines l ₁ to l ₁₂ are formed radially from this point q every 30 °. In addition,
The angle is not limited to 30 °, and it is also possible to form straight lines at unequal angles.

Next, a histogram is created on each straight line l (ST11
3). 2 (a), (b) and (c) are schematic diagrams for explaining an example of this histogram. Next, it is determined whether the minimum point can be extracted from the histogram obtained in ST113,
If this determination is YES, the process branches to ST115, and if NO, the process branches to ST116 (ST114). In ST115, it is further determined whether or not there is one minimum point, and if YES, the process branches to ST117, and if NO (when there are two or more minimum points), ST118.
Branch to.

FIG. 2 (a) shows a case where there is no minimum point in the histogram. In this case, the process proceeds to ST116 and the point intersecting the threshold value n _th is set as the boundary point r. FIG. 2B shows the case where there is one minimum point X, but in this case, ST117
Then, the one minimum point is determined as the boundary point r. Second
FIG. 7C shows the case where there are _two local minimum points X ₁ and X _{2. In} this case, the procedure proceeds to ST118, and the local minimum point X ₁ closest to the highest numerical value point q is set as the boundary point r. .

In ST119, it is determined whether or not the boundary points r have been determined for all straight lines l, and if this determination is NO, ST113
A histogram is created for the next straight line, and if YES, the process returns to the main routine of FIG. 4 (a) and proceeds to the process of ST12. In FIG. 1, the final fraction was calculated. Boundary points r _{1 to} r for each straight line l _{1 to} l _12
Twelve are shown.

In ST12, the region obtained by connecting the boundary points r obtained in ST11 with a straight line is determined as the final fraction. In the case of FIG. 1, the area B ′ obtained by connecting the boundary points r _{1 to} r ₁₂ with a straight line is determined as the final fraction. This final fraction B'is more compatible with the distribution of lymphocytes than fraction B obtained in ST10. In addition, the processing time of ST11 and ST12 can be shortened as compared with the contour tracking method.

For the data that belongs to the final fraction determined in ST12,
Further, fluorescence characteristic analysis is performed (ST13). In this fluorescence characteristic analysis, for example, a histogram is created for each of the green fluorescence intensity I _g and the red fluorescence intensity I _r , and the positive rate is calculated. The result of this fluorescence characteristic analysis is displayed on the CRT 23 (ST14) and printed out from the printer 24 (ST
15).

In ST16, it is determined whether or not there is a sample to be measured.
If this determination is YES, the procedure returns to ST2 to measure the next sample, and if NO, the measurement is terminated.

In the above description, the analysis of lymphocyte subsets is taken as an example, but the present invention is not limited to this, and analysis of various cell (or particle equivalent thereto), for example, red blood cells, reticulocytes, platelets, etc. It is widely applicable to. Also, the parameters to be used are 4 of forward scattered light intensity, 90 ° scattered light intensity, green fluorescence intensity, and red fluorescence intensity.
It is not limited to types. Furthermore, the present invention can be applied to a cell analyzer using the window method, and in this case, the final fraction is determined in the window.

(G) Effect of the Invention As described above, the cell analysis device of the present invention has the highest numerical value point on the cytogram relating to the fractionation of the cell population for the cell light information collected by the cell light information collecting means. The highest degree numerical point extracting means for extracting, on the cytogram, a straight line forming means for forming a straight line on the radiation from the highest degree numerical point extracted by the highest degree numerical point extracting means, and this straight line forming means Boundary point extraction means for extracting the boundary points from the histogram on each of the formed straight lines,
An area formed by connecting the boundary points extracted by the boundary point extracting means is defined as a final fraction, and second cell light information collecting means for collecting cell light information of the target cell population based on the final fraction. Since it is provided, it has an advantage that a fraction more suitable for the target cell population can be determined in a short processing time and the accuracy of analysis can be improved.

[Brief description of drawings]

FIG. 1 is a diagram showing a main part of a cytogram displayed in contour lines for explaining the final fraction calculation of the cell analyzer according to one embodiment of the present invention, FIGS. 2 (a), 2 (b) and FIG. 2 (c) is a schematic diagram of a histogram for explaining the final fraction calculation of the cell analyzer, FIG. 3 is a block diagram for explaining the configuration of the cell analyzer, and FIG. 4 (a) is , A flow chart for explaining the overall operation of the cell analyzer, FIG. 4 (b), a flow chart for explaining the final fraction calculation of the cell analyzer, FIGS. 5 (a) and 5 (b). Is a histogram of 90 ° scattered light intensity and forward scattered light intensity for explaining the fraction extraction calculation of the cell analyzer, and FIG. 6 is an example of a cytogram of forward scattered light intensity and 90 ° scattered light intensity. Fig. 7 (a) and Fig. 7 (b) show the 90 ° scattered light intensity and forward scattering, respectively. Is a diagram showing an example of a histogram of intensity. 10: Flow cell, 14: Laser, 15a ・ 15b ・ 15c ・ 15d: Photodetector, 21: MPU, 23: CRT, 24: Printer.

Claims (1)

[Claims] 1. A cell suspension flowing in a flow cell is irradiated with a light beam to extract cell light information of a target cell population from cell light information obtained from each cell, and the cell light information collecting means collects the cell light information. In the cell analysis device for analysis processing, the cell light information collected by the cell light information collecting means, the highest degree numerical point extraction means for extracting the highest degree numerical point on the cytogram relating to the fractionation of the cell population, On the cytogram, straight line forming means for forming a straight line on the radial line from the highest degree numerical point extracted by the highest degree numerical point extracting means, and a histogram for each straight line formed by this straight line forming means , The boundary point extraction means for extracting the boundary points and the region formed by connecting the boundary points extracted by the boundary point extraction means are defined as the final fraction, and the target cell collection is performed based on this final fraction. And a second cell light information collecting means for collecting cell light information of the group.