JP2893613B2 - Micronucleus cell analysis method and micronucleus cell analyzer - Google Patents

Description

The present invention relates to a method and an apparatus for analyzing micronucleated cells by applying flow cytometry.

(B) Prior art Micronucleus cells have small nuclei generated by natural or artificial processing other than normal nuclei, and are formed when chromosomal fragments or chromosomes due to divisional delay cannot move to both poles. The frequency of micronucleated cells is used as a measure of chromosomal abnormalities based on radiation damage and environmental factors. In the conventional micronucleus cell analysis method, a sample, for example, a bone marrow fluid is smeared on a slide glass, Giemsa staining, DAPI staining, or the like is performed, and the micronucleus cells are counted by visual observation (microscope) using a microscope. Was.

(C) Problems to be Solved by the Invention The above-described conventional method for analyzing micronucleated cells has a problem that it is inefficient and requires time and labor because counting is performed using a microscope. Also, if the micronucleus is human red blood cells,
They have various sizes from about 0.5 μm to about 2 μm, and there is a problem that their discrimination requires skill.

The present invention has been made in view of the above, and an object of the present invention is to provide a micronucleus cell analysis method and apparatus capable of analyzing micronucleus cells at high speed without labor and without requiring skill.

(D) Means for Solving the Problems In order to solve the above problems, the micronucleus cell analysis method according to the first aspect has a configuration listed in the following items i to v.

i: cells or bone marrow cells in peripheral blood are stained with acridine orange, ii: the stained cells or the like are allowed to flow in a thin stream, iii: fluorescence of acridine orange is applied to cells or the like flowing in the stream. By irradiating the exciting light beam, at least the fluorescence peak intensity is detected as the cell light information among the signal light intensity generated from the cells, etc. And v: analyzing micronucleus cells based on the cell light information of the selected micronucleus cells.

Further, the micronucleus cell analyzer according to the second aspect has the following I-
It has the configuration listed in VI.

I: a flow cell through which a suspension such as cells stained with acridine orange flows, II: a light source that irradiates a cell or the like flowing through the flow cell with a light beam, and III: each cell or the like irradiated with this light beam. about,
Cell light information detection means for detecting cell light information including at least the fluorescence peak intensity; IV: From the cell light information detected by the cell light information detection means, based on the fluorescence peak intensity, cell light information of micronucleus cells A micronucleus cell light information sorting means for sorting from cell light information of other cells, etc., V: an analysis processing means for analyzing and processing the micronucleus cell light information selected by the micronucleus cell light information sorting means, VI: output means for outputting the processing result of the analysis processing means.

(E) Function Flow cytometry is a technique in which cells and the like are caused to flow in a fine liquid flow, and a light beam is applied to each of the cells flowing in a line by a hydrodynamic focusing effect, thereby generating scattered light generated from the cells. And instantaneously measure the intensity of fluorescence, ie, cell light information, and analyze the cells. It is possible to analyze a large number of cells at high speed and with high accuracy.

In order to apply this flow cytometry to the analysis of micronucleated cells, it is necessary to stain the micronucleated cells so that they can be distinguished from other cells and the like. Therefore, the present inventors have focused on acridine orange.

Acridine orange has long been used for cell biochemical research, and has been used for the qualitative and quantitative analysis of intracellular DNA and RNA, especially because of its characteristic of simultaneously dyeing DNA and RNA. I have. This is due to the fact that two types of AO bonds are found in nucleic acids, which bind to double-stranded nucleic acids by intercalation, and which bind to single-stranded nucleic acids electrostatically. The 488 nm excitation light of the laser produces green fluorescence with a wavelength of 530 nm, and the latter produces the same excitation light producing red fluorescence with a wavelength of 640 nm.

Normally, in vivo, DNA almost has a double helix structure, so that DNA is detected as green fluorescence by the DNA-AO complex, while RNA exists in either a single-stranded or double-stranded form. Needs to be modified to a single-chain structure. For this, a chelating agent of a divalent cation such as Citrate or EDTA, particularly Mg ²⁺ is often used. Therefore, DNA emits green fluorescence in a double strand and RNA emits red fluorescence in a single strand, so that DNA and RNA can be measured simultaneously by detecting each.

However, acridine orange slightly stains not only DNA and RNA but also intracellular reticulocytes. However, when reticulocytes are stained, FIG. 2 (b)
As shown in (c), it is dyed as a whole or mottled. On the other hand, in the case of micronuclei as shown in FIG. 2A, in many cases, strong fluorescence is generated in a small area. Therefore, in the case of micronuclei, the peak of fluorescence
As shown in FIG. 3A, a sharp large peak occurs, but in the case of reticulocytes, the whole or mottled portions are as shown in FIGS. By detecting, cell light information of micronucleus cells can be selected from cell light information of other cells or the like, and analysis can be performed.

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

FIG. 4 is a diagram showing a configuration of the cell analyzer of the embodiment. Reference numeral 2 denotes an autosampler, and its sample rack 2a
Are loaded with a plurality of sample containers 3,. The sample rack 2a is driven by a drive mechanism (not shown),
The designated sample is located immediately below the sample suction tube 4.
Further, the autosampler 2 is provided with a shaking mechanism and a cooling mechanism (not shown), which shakes the sample at regular intervals, and lowers the temperature of the sample in the loaded sample containers 3,. 1010 ° C.).

The sample suction tube 4 is connected to one port of a three-way valve 5. The other two ports of the three-way valve 5
The sample feeding tubes 6a and 6b are connected respectively, and the sample feeding tube 6a and the sample suction tube 4 or the sample feeding tubes 6a and 6b can be communicated. 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 liquid sending tube 6b is opened inside the sheath liquid sending 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 quartz glass or the like, a sheath flow is formed in the internal flow channel 10a, and cells and the like in the sample flow in a line on the central axis of the flow channel 10a due to a hydrodynamic focusing effect. It is.

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. Note that the micronucleus 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. 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, an argon ion laser 14,
Photodetectors 15a, 15b, 15c and 15d are provided. The laser beam (λ = 488 nm) 1 from the argon ion laser 14 is
Irradiation is performed on cells and the like flowing through the flow channel 10a. At this time, in the flow channel 10a, the laser beam l
Is preferably narrowed to 5 μm or less with respect to the flowing direction of the cell suspension in order to capture the fluorescence peak intensity.

Signal light is generated from the cells irradiated with the laser beam l, and those in the forward direction are those as forward scattered light,
The light is collected by the lens 16a and is incident on the photodetector 15a. 17
Is a beam blocker for preventing the laser beam 1 from directly entering the photodetector 15a.

On the other hand, the signal light in the 90 ° direction from the cell is collected by the lens 16b. A part of this signal light is reflected by the dichroic mirror 18a, and the light detector 15b for detecting 90 ° scattered light is used.
Incident on. 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 light transmitted through the dichroic mirror 18b is transmitted through the filter 19b and received by the photodetector 15d for red fluorescence. For example, a photodiode is applied to the photodetector 15a for forward scattered light, and a photomultiplier is applied to the other detectors 15b, 15c, and 15d.

The light receiving signals of the photodetectors 15a, 15b, 15c, 15d are amplified by a signal processing circuit 20, then converted into digital signals by an analog / digital (A / D) converter 21, and taken into the MPU 22.

A detailed block diagram of the signal processing circuit 20 is shown in FIG. The signal processing circuit unit 20 includes a signal processing circuit 20 for forward scattered light intensity.
a, 90 ° scattered light intensity signal processing circuit 20b, green fluorescent peak intensity signal processing circuit 20c, green fluorescent area intensity signal processing circuit 20c ′ red fluorescent peak intensity signal processing circuit 20d, red fluorescent area intensity signal processing It is constituted by a circuit 20d '.

The signal processing circuit for forward scattered light intensity 20a and the signal processing circuit for 90 ° scattered light intensity 20b are each configured by a preamplifier, an integrator, a linear (Lin) amplifier, and a peak hold circuit, and their outputs I ₀ , I ₉₀ is converted into a digital signal by the A / D converter 21 and taken into the MPU 22.

The green fluorescence peak intensity signal processing circuit 20c and the red fluorescence peak intensity signal processing circuit 20d are both configured by a preamplifier, a linear amplifier, and a peak hold _circuit.The outputs I _gp and I _rp are respectively output by the A / D converter 21. It is converted into a digital signal and taken into the MPU22.

The green fluorescence area intensity signal processing circuit 20c 'includes an integrator that integrates the output of the preamplifier of the green fluorescence peak intensity signal processing circuit 20c, a linear amplifier that amplifies the output of the integrator, and a logarithmic (log) amplifier. , And a peak hold circuit for peak-holding the output of each of the linear amplifier and the logarithmic amplifier. The output of each peak hold circuit is converted into a digital signal by the A / D converter 21 as a green fluorescent area intensity (linear) I _ga and a green fluorescent intensity (log) I _ga ′, and is taken into the MPU 22. The red fluorescent area intensity signal processing circuit 20d 'is exactly the same, and the two outputs, the red fluorescent area intensity (linear) I _ra and the red fluorescent intensity (log) I _ga ', are converted into digital signals, which are sent to the MPU 20. It is captured.

The MPU 22 has a function of selecting cell light information of a target cell population (in this example, red blood cells) as a stage prior to cell light information obtained from a sample based on the forward scattered light intensity I ₀ and the 90 ° scattered light intensity I _90. A function of selecting and analyzing cell light information of micronucleus cells from cell light information of a target cell population based on the fluorescence peak intensity and the fluorescence area intensity, and also controls the sample pump 7, the liquid supply pump 9 and the autosampler 2. It has functions and the like.

A floppy disk drive 23, a keyboard 24, a CRT 25, and a printer 26 are connected to the MPU 22. The floppy disk drive 23 is for storing measurement conditions (protocols), measurement data, and the like on a floppy disk. The keyboard 24 is for inputting selection / setting of a protocol or other commands to the MPU 22. The CRT 25 is for monitoring the measurement, and the printer 26 is for printing out a processing result of the MPU 22, for example, a cytogram or a histogram.

Next, the operation of analyzing the micronucleated red blood cells by the micronucleated cell analyzer of the embodiment will be described.

First, preparation of a sample will be described. This sample is mixed with 10 μl of peripheral blood and 2 ml of a staining solution, and left (incubated) at room temperature for 3 to 5 minutes to stain micronuclei. This staining solution is
For example, a commercially available acridine orange staining solution (manufactured by Ortho Diagnostic Systems) can be used.

As another method for preparing a sample, for example, a 0.1% aqueous solution of acridine orange is prepared, and 1 volume of this solution is diluted with 99 volumes of a phosphate buffer solution (1/15 M, pH 6.8). There is also a method of adding 10 μm of peripheral blood to 2 ml and incubating at room temperature for 3 to 5 minutes.

When the power of the micronucleus cell analyzer is turned on, a program is read into the MPU 22 from a ROM (not shown), and the system starts up. Then, a protocol suitable for the sample to be measured is selected and set [Step (hereinafter referred to as ST)].
1, see FIG. 6). This protocol uses photodetectors 15a, 1
The content includes measurement conditions such as detector gains and correction calculations for 5b, 15c, and 15d.

Next, in ST2, the sample mixed with the staining solution by the method described above is set in the autosampler 2, and the measurement is started. When a predetermined time has elapsed after the incubation of the sample (for 3 to 5 minutes), the sample rack 2a is driven, and the sample container 3 containing the sample is located immediately below the sample suction tube 4. Then, the three-way valve 5 is switched so as to communicate the sample suction tube 4 and the sample feeding tube 6a, the sample suction tube 4 descends into the sample container 3, the sample pump 7 is driven to the suction side, and the sample Is sucked into the sample feeding tube 6a.

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 the cells flow in a line on the central axis of the flow channel 10a due to the hydrodynamic focusing effect. Forward scattered light intensity I ₀ , 90 ° scattered light intensity I ₉₀ , green peak intensity I _gp , green area intensity (linear) I _ga , green area intensity generated when each of these cells crosses the laser beam 1 (Log) _Iga ', red peak intensity _Irp , red area intensity (linear) _Ira , and red area intensity (log) _Ira ' are measured. These cell light information is sequentially stored on a floppy disk or the like as needed.

In the measurement end ST3, a predetermined operation is performed on the cell light information of the analysis area A in order to select the cell light information of red blood cells from the cell light information of other cells such as white blood cells as a pre-stage of the fluorescence characteristic analysis. The analysis region A is set on the forward scattered light intensity I ₀ , 90 ° scattered light intensity I ₉₀ cytogram, as shown in FIG. 1 (a). In FIG. 1 (a), b, c, d
The distributions of red blood cells, lymphocytes, monocytes, and granulocytes are shown, respectively, and the analysis area A is set so as to surround the distribution b of red blood cells and lymphocytes. This setting can be manually set by a user using a mouse, a cursor key, or the like, or an analysis area can be automatically set (so-called auto trigger). The cell light information belonging to the analysis area A is selected from the measured cell light information.

In ST4, it is determined whether or not the analysis area A is reset. When the determination is NO, the process branches to ST6, and when the determination is YES, the process branches to ST5, a new analysis region is set and recalculation is performed, and then the process proceeds to ST6.

In ST6, fluorescence characteristic analysis is performed. In this fluorescence characteristic analysis, first, an analysis area B is set on the cytogram of the green fluorescence peak intensity I _gp and the green fluorescence area intensity (log) I _ga ′ (see FIG. 1B). In this cytogram, b ₁ , b ₂ , and b ₃ are red blood cells, red blood cell micronucleus cells,
The distribution of lymphocytes is shown. Analysis area B is
The red blood cells are set so as to surround the distribution b ₂ micronucleus cell, the settings manually, an automatic one possible.

Acridine orange stains not only micronuclei but also reticulocytes. However, in the case of micronuclei, as shown in FIG. 2 (a), strong fluorescence is generated in a small area, so that the signal of the photodetector 15c (similarly, 15d) is as shown in FIG. 3 (a). It becomes a peak shape. On the other hand, in the case of reticulocytes, the whole red blood cells are stained (see FIG. 2 (b)) or spotted (see FIG. 2 (c)), so that the photodetectors 15c (15d) The signals are respectively shown in FIG.
As shown in (c), the waveform is such that gentle low peaks and small peaks continue. For this reason, it is possible to separate erythrocytes and erythrocyte micronuclei by the green fluorescence peak intensity _Igp .

Since acridine orange is usually reacted with whole blood, the nuclei of leukocytes are also stained. However,
The size and the like of the endolymphocytes of red blood cells are similar to those of red blood cells, and the scattered light intensities I ₀ and I ₉₀ are also similar. Therefore, when the cell light information is selected by setting the analysis area A, there is a possibility that cell light information of lymphocytes may be mixed with red blood cells at a ratio of 500: 1 to 1000: 1.

However, since lymphocytes have both DNA and RNA and their nuclei mainly emit fluorescence, green fluorescence and red fluorescence are strongly detected in a wide range, and thus can be easily distinguished from erythroid micronucleus cells. That is, in the case of the embodiment, the green fluorescence peak intensity I _gp and the green fluorescence area intensity (log) I _ga ′
On cytogram, it can be selected by separating the distribution b ₃ of the red blood micronucleus cell distribution b ₂ and lymphocytes.

Further, for the cell light information selected in the analysis region B, a red fluorescence peak intensity I _rp and a red fluorescence area intensity (log) I _ra ′ cytogram are created (see FIG. 1 (c)). In this cytogram on, but b ₂₁ shows a distribution of red blood micronucleus cells, b ₂₂ are platelets is the distribution of Les cyclamate site. Therefore, if counting the number of cells belonging to b _21,
It is possible to know the exact number of micronucleated cells.

In ST7, the result of the fluorescence characteristic analysis is displayed on the CRT 25, and in ST8, the result is printed out by the printer 26. In ST9, it is determined whether or not there is a sample to be measured. If YES, the process returns to ST1, and if NO, the analysis ends. When returning to ST1, the operation is performed in the same manner as described above.

In this embodiment, 6 out of 8 types of cell light information are used.
The case where micronucleated cells are analyzed using types is shown as an example,
The type and number of cell light information to be used are not limited to those described above, and the procedure of the fluorescence characteristic analysis is not limited to the embodiment, and the setting can be changed as appropriate.

Further, in this example, the analysis of erythrocyte micronucleus cells has been described. However, the present invention can be similarly applied to the analysis of leukocyte micronucleus cells, and can be applied not only to humans but also to micronucleus cells of other animal species. is there.

(G) Effects of the Invention As described above, the micronucleus cell analysis method of the first aspect stains cells or the like or bone marrow cells in peripheral blood with acridine orange, and stains the stained cells or the like with a thin liquid flow. Irradiating the cells and the like flowing in this liquid flow with a light beam for exciting the fluorescence of acridine orange, and detecting at least the fluorescence peak intensity as the cell light information among the signal light intensities generated from the cells and the like. Based on the fluorescence peak intensity, cell light information of micronucleus cells is selected from cell light information of other cells and the like, and micronucleated cells are analyzed based on the cell light information of the selected micronucleus cells. It has the advantage of being able to analyze micronucleated cells at high speed, without labor and without skill.

A micronucleus cell analyzer according to a second aspect of the present invention includes a flow cell through which a suspension such as cells stained with acridine orange flows, a light source that irradiates a cell or the like flowing through the flow cell with a light beam, For each of the irradiated cells, for example, cell light information detecting means for detecting cell light information including at least the fluorescence peak intensity, and based on the cell light information detected by the cell light information detection means, based on the fluorescence peak intensity , Micronucleus cell light information selection means for selecting cell light information of micronucleus cells from cell light information of other cells, etc., and analysis processing of micronucleus cell light information selected by this micronucleus cell light information selection means Analysis means, and an output means for outputting the processing result of the analysis processing means. Therefore, there is an advantage that the analysis of the micronucleus cell according to the first aspect can be easily performed. .

[Brief description of the drawings]

FIGS. 1 (a), 1 (b), and 1 (c) are diagrams each schematically showing a cytogram illustrating a procedure for analyzing erythrocyte micronucleus cells according to one embodiment of the present invention. , FIG. 2 (a), FIG. 2 (b) and FIG. 2 (c) schematically show the state of red blood cell staining with acridine orange, respectively, FIG. 3 (a), FIG. 3 (b) ) And FIG. 3 (c)
Correspond to FIGS. 2 (a), 2 (b) and 2 (c), respectively, and explain the light reception signal of a fluorescence photodetector for red blood cells stained with acridine orange. FIG. 4 is a block diagram illustrating the configuration of a micronucleus cell analyzer applied to this embodiment, and FIG. 5 is a block illustrating details of a light reception signal processing circuit unit of the micronucleus cell analyzer. FIG. 6 and FIG. 6 are flowcharts for explaining the operation of the micronucleus cell analyzer. 10: flow cell, 14: argon ion laser, 15a, 15b, 15c, 15d: photodetector, 20: light receiving signal processing circuit, 22: MPU, 25: CRT, 26: printer.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁶ , DB name) G01N 33/48-33/98

Claims (2)

(57) [Claims] 1. A cell or the like in peripheral blood or a bone marrow cell is stained with acridine orange, and the stained cells or the like are flowed in a thin liquid flow, and the fluorescence of acridine orange is excited in the cells or the like flowing in the liquid flow. Of the signal light generated from the cells and the like, at least the fluorescence peak intensity is detected as the cell light information, and based on the fluorescence peak intensity, the cell light information of the micronucleus cell is converted to other cells and the like. A micronucleus cell analysis method for sorting micronuclei cells based on the cell light information of the selected micronucleus cells. 2. A flow cell through which a suspension such as cells stained with acridine orange flows, a light source for irradiating a cell or the like flowing through the flow cell with a light beam, and each cell or the like irradiated with the light beam. A cell light information detecting means for detecting cell light information including at least the fluorescence peak intensity, and a cell light information detected by the cell light information detecting means,
Micronucleus cell light information selection means for selecting cell light information of micronucleus cells from cell light information of other cells based on the fluorescence peak intensity, micronuclei selected by the micronucleus cell light information selection means A micronucleus cell analyzer comprising: an analysis processing means for analyzing and processing cell light information; and an output means for outputting a processing result of the analysis processing means.