JPH04140661A - Method and device for analyzing degree of fluorescent deflection of cell - Google Patents

Abstract

PURPOSE:To enable accuracy of analysis to be improved by analyzing degree of fluorescent deflection based on strength of deflection constituent of fluorescence of information of a selected target cell group. CONSTITUTION:A laser beam l from a laser light source 2 is emitted to a cell which flows within a flow channel 1a through a lambda/2 plate 3. A signal light is generated from the cell and a signal light l1 in its internal forward direction is collected at a lens 5 as a forward scattering light and enters a light detector 13a. Also, a beam blocker 4 prevents a beam l$0 from entering the detector 13a directly. On the other hand, a signal light l2 in a direction of 90 degrees from the cell is collected by a lens 6, its one part is reflected by a dichroic mirror 7, and then it enters a light detector 13b for detecting 90-degree scatter ing light a filter 9 allows light with wavelength from the light source 2 to be transmitted and prevents fluorescent constituent from the cell. Then, signal light which is transmitted through the mirror 7 partially enters the dichroic mirror 8 and is selected by a filter 11, and then it enters a light detector 13e for detecting fluorescence of PE.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application The present invention relates to a method and apparatus for analyzing the degree of fluorescence polarization of cells using flow cytometry.

(B) Conventional technology The fluorescence polarization degree P of a cell is related to the viscosity and fluidity of the cell membrane and cytoplasm, and can provide information on cell activation and juvenile development. The possibility of cancer diagnosis based on the degree of fluorescence polarization of lymphocytes has also been suggested.

(Cerek, L, Cerek, B, Eur, J,
Cancer 1977Vol1. ．． 13, P2O3-
915).

To explain the degree of fluorescence polarization P with reference to FIG. 5, C is a cell stained with a fluorescent dye and is excited by excitation light traveling in the Z-axis direction. ■Evening shows polarized light in the X direction, and Bi1 shows polarized light in the X direction.

In order to reduce interference caused by the excitation light, detection is usually performed in a direction perpendicular to the traveling direction of the excitation light, in the case of FIG. 5, from the X direction. ■11.I indicates the polarization component of fluorescence, and IIl is the polarization direction of excitation light I! indicates a polarized component parallel to , and I indicates a perpendicular parallel component. Fluorescence polarization degree P
is the polarization direction of the excitation light 1. From I, , I when , it can be expressed by the following equation (1).

P= (I,,-1,)/(1,,+1)...(
1) On the other hand, the polarization direction of the excitation light is Bi! When , the polarization direction of fluorescence is 'II, ■, the polarization direction of excitation light is 1゛! The angle of inclination is the same as that of , and both IIl and I have a polarization direction perpendicular to ■゛J. At this time, regardless of the degree of fluorescence polarization of cell C,
IIl and ■ indicate the same value. This development is used to adjust the fluorescence detection sensitivities in both light directions to be the same.

In order to perform an actual analysis, for example, when analyzing the degree of fluorescence polarization of lymphocytes, blood is collected from the subject, and this blood is then collected into the lymphocytes using specific gravity centrifugation or the phagocytic ability of neutrophils. The spheres are sorted and fluorescently labeled according to the measurement purpose.

The suspension of lymphocytes thus obtained is placed in a cuvette, and the polarized light component is measured using the measuring device shown in FIG. This cuvette 31 is made of an optically transparent material such as quartz, and is irradiated with excitation light 33 from a light source 32. Note that the polarization direction of this excitation light 33 is perpendicular to the plane of FIG. 6 due to the λ/2 plate 34 (hereinafter, the polarization direction perpendicular to the plane of the paper is indicated by the symbol "○").

The light 40 from the irradiation area 31a of the excitation light 33 includes scattered light, a fluorescence polarized component perpendicular to the paper surface, and a polarization component parallel to the paper surface (hereinafter, the polarization direction perpendicular to the paper surface is referred to as "1" or "1").
). This light 40 is condensed by a condenser lens 35, and fluorescence is selected by a fluorescence selection filter 36. Furthermore, the polarization beam splitter 37 separates the light into a polarization component perpendicular to the plane of the paper and a polarization component parallel to the plane of the paper, which are converted into electrical signals by the photodetectors 38a and 38b. Polarization degree P
is calculated.

Another method is to measure the lymphocyte suspension using a flow cytometer equipped with a fluorescence polarization degree detection function.

(C) Problems to be Solved by the Invention The conventional measurement of the degree of fluorescence polarization described above requires an operation to select a target cell population, for example, to select lymphocytes from blood. However, the selection of lymphocytes requires complicated and delicate operations, which poses the problem of time and skill.

In addition, such sorting has the problem that it is difficult to increase the purity of the target cell population, and that it often affects cell activity, making it difficult to obtain accurate data.

Furthermore, it is difficult to finely sort cell populations, for example, to sort subgroups of lymphocytes (B cells, Tt4 cells, etc.), and there are problems in that it is difficult to analyze the degree of fluorescence polarization for each subgroup.

The present invention has been made in view of the above, and aims to provide a cell fluorescence polarization analysis method and an analysis device that allow easy sample preparation, high analytical accuracy, and allow for finely selecting and analyzing cell populations. It is said that

(d) Means and action for solving the problem In order to solve the above problem, the cell fluorescence polarization degree analysis method of the first claim includes:
Cells in a sample are stained with at least a fluorescent dye related to polarization analysis, the sample is flowed into a thin liquid stream, the flowing cells are irradiated with a light beam to excite the fluorescence of the fluorescent dye, and for each cell, Cell light information consisting of the intensity of the polarized component of the fluorescence and other parameters is detected, and by using the other parameters of the cell light information, the cell light information of the target cell population is compared with the cell light information of other cell populations. The degree of fluorescence polarization of the selected target cell population is analyzed based on the polarization component intensity of the fluorescence of cell optical information of the selected target cell population.

In the cell fluorescence polarization analysis method of the present invention, after measuring cell light information for all cells in a sample, other parameters such as forward scattered light intensity, 90° scattered light intensity, fluorescence intensity (fluorescence polarization analysis) are measured. This method uses the intensity of fluorescence caused by other fluorescent dyes that are not directly involved in the method to select the cell light information of the target cell population from the cell light information of other cell populations. Therefore, there is no need to actually select the target cell population from the sample, making it easier to prepare the sample, improving the accuracy of selecting the target cell population, and enabling accurate analysis.

Furthermore, staining with the other fluorescent dyes described above was performed using fluorescent dyes.
By reacting the identified monoclonal antibody with cells in a sample, the specificity of the monoclonal antibody makes it possible to sort out detailed cell populations, such as lymphocytes T cells, B cells, etc., by cell optical information. The degree of fluorescence polarization for these fine cell populations can be analyzed.

On the other hand, the cell fluorescence polarization analyzer according to the second aspect includes at least a flow cell in which a suspension of cells stained with a fluorescent dye related to polarization analysis is flowed, and a cell flowing through the flow cell that is provided with the fluorescent dye. a light source that irradiates a light beam that excites fluorescence; a cell light information detection means that detects cell light information consisting of the polarization component intensity of the fluorescence and other parameters for each cell irradiated with the light beam; Cell light information selection means for selecting cell light information of a target cell population from cell light information of other cell populations using the other parameters obtained by the cell light information detection means, and this cell light information selection means A cell fluorescence polarization degree analysis means for analyzing the degree of fluorescence polarization of the target cell population based on the polarization component intensity of the fluorescence of the cell light information of the target cell population selected by the method, and outputting the analysis results of this cell fluorescence polarization degree analysis means. The cell fluorescence polarization degree analysis method according to the first aspect can be easily carried out.

(E) Embodiment An embodiment of the present invention will be described below with reference to FIGS. 1 to 4.

FIG. 2 is a block diagram illustrating the configuration of the cell fluorescence polarization analyzer according to the embodiment. 1 is a flow cell, which is made of quartz glass or the like. At the center of flow cell 1,
A flow channel 1a is formed, and cells in the sample are flowed along with the sheath liquid in a direction perpendicular to the plane of the paper in FIG. These cells flow in a line on the central axis of the flow channel 1a due to the hydrodynamic focusing effect. In addition,
The liquid feeding system that feeds the sheath liquid and sample to the flow cell 1 is as follows:
It is omitted because it is not an essential part of the present invention (Patent Application No. 1983-1
59866).

In this embodiment, the laser light source 2 is a He-Cd laser (oscillation wavelength: 421 nm) or an Ar laser (oscillation wavelength: 45 nm).
8 nm or 488 nm). Laser light source 2
The laser beam 1 passes through a λ/2 plate 3 for rotating the polarization direction perpendicularly or parallel to the plane of FIG. 2, and is irradiated onto cells flowing in the flow chamber 1a.

Signal light is generated from cells, but the signal light is directed inside!
The light is focused by the lens 5 as forward scattered light, and is incident on the photodetector 13a. 4 is Leather Beam! . This is a beam blocker that prevents the light from directly entering the photodetector 13a.

On the other hand, the signal light 12 in the 90° direction from the cell is focused by the lens 6. A portion of the signal light 12 is reflected by the guichroic mirror 7 and enters a photodetector 13b for detecting 90° scattered light. Reference numeral 9 denotes a filter that transmits light having the wavelength of the laser light source 2 and blocks fluorescent components from cells.

A portion of the signal light transmitted through the guichroic mirror 7 enters another guichroic mirror 8. This dichroic ink mirror 8 is used for fluorescence polarization analysis of lymphocytes (described later) from the fluorescent dye PE (510 nm).
) and fluorescein fluorescence (with a peak at 575
This is a device that is used to separate substances that have a peak at nm.

The signal light transmitted through the dichroic mirror 8 is transmitted to the PE
This fluorescence is sorted by the filter 11 and P
E enters the photodetector 1.3 e for fluorescence detection.

The fluorescent light reflected by the dichroic mirror 8 passes through a filter 10 that transmits fluorescein fluorescence, and enters a polarizing beam splitter, in this embodiment, a Glan-Thompson prism 12, where the polarized light component perpendicular to the plane of FIG. The perpendicular polarized light component and the parallel polarized light component are incident on photodetectors 13d and 13c, respectively.

In this embodiment, the photodetector 13a for forward scattered light includes a photodiode, and the other photodetectors 13b, 13c, 1
．． The photodetectors 13c and 13d are equipped with a fluorescence detection sensitivity correction circuit 14.
connected to. From the flow cell 1 to the photodetector 1. Jc, 1
The transmission, reflection, and light detection characteristics of an optical system up to 3D are generally not the same for a polarization component perpendicular to the plane of FIG. 2 and a polarization component parallel to it. Therefore, in the fluorescence detection sensitivity correction circuit 14,
Correction is made so that the photodetectors 13c and 13d generate signal outputs of the same magnitude for polarized light components of the same intensity emitted from the cells.

Photodetector 13C113 corrected by this correction circuit 14
The signal output of d and the signal outputs of the photodetectors 13a, 13b, and 13e are amplified by a signal processing circuit section 15, then digitally converted by an analog/digital (A/D) converter 16, and taken into the MPU 17. It will be done. Signal processing circuit section 15
Although the details are not shown, each photodetector 1.3a-13e
The signal output is amplified by a preamplifier, integrated by an integrator, further amplified by a log (or linear) amplifier, held at its peak by a peak bold circuit, and output to the A/D converter 16.

The MPU 17 has a list memory function to store optical information from cells, a function related to auto trigger, a function to create a histogram and a cytogram for one or two parameters of the cell optical information, and other functions to control the liquid delivery system. We are prepared.

A keyboard 18, a CRT 19, and a printer 20 are connected to the MPU 17. The keyboard 18 is used to input various commands and data. Note that if a mouse is also used as an input means, the operation will be easier. The CRT 19 and printer 20 have the above histogram,
It is used to display and print cytograms and analysis results.

Next, the operation of the cell fluorescence polarization analyzer according to the embodiment will be explained.

In the following explanation, fluorescence polarization analysis of lymphoid T cells will be taken as an example. As a fluorescent dye for fluorescence polarization analysis,
Fluorecein is used, and P E (phycho erythrin) is applied as a fluorescent dye to separate cell optical information of T cells and B cells.

and forward scattered light intensity I for each cell. , 90° upper scattered light intensity I,. , PE fluorescence intensity IF, fluorescein perpendicular polarization component intensity 1, parallel polarization component intensity I, and forward scattered light intensity ■. , 90° scattered light intensity I,. to sort the cell light information of lymphocytes using
Cell light information of T cells is further selected by PE fluorescence intensity IP.

Before measurement, the fluorescence detection sensitivity correction circuit 14 is adjusted. First, the laser beam that enters flow cell 1!
. λ so that the polarization direction of is parallel to the plane of the second drawing.
/2 Adjust the rotation of plate 3.

Next, latex particles labeled with fluorescein isothiocyanate (FITC) are passed through the flow cell 1. The signal outputs of the photodetectors 13c and 13d are monitored with an oscilloscope (not shown), and the pulse heights are made to be the same.
Adjust the amplifier gain of the fluorescence detection sensitivity correction circuit. After adjustment, the laser beam enters flow cell 1! . λ/ so that the polarization direction of is perpendicular to the plane of the paper as shown in Figure 2.
Set by rotating the 2nd plate 3. Note that this adjustment does not need to be performed at every measurement.

Next, preparation of the sample will be explained. First, whole blood is washed with a phosphate buffer and serum components are removed. Next, PE-labeled monoclonal antibody 0BK7 is added, and after reacting for several minutes, it is hemolyzed and washed. Furthermore, F.D.
Add A (fluorecein diaietate). FDA permeates cell membranes and is converted to fluorescein by intracellular enzymes.

After reacting in this manner for several minutes, measurements are taken.

First, the sample is sucked into a liquid feeding system (not shown) and flows through the flow channel la along with the sheath liquid.

Cells in the sample flow in a line on the central axis of the flow channel 1a. Each cell is a laser beam! . Signal light when crossing! ! The forward scattered light intensity I is generated. , 90° scattered light intensity■,. , PE fluorescence intensity I2, vertical polarization component intensity I, and parallel polarization component intensity I are measured [Step (hereinafter referred to as ST) 1, see FIG. 1]. The cell light information thus measured is stored in the memory of the MPU 17 (list memory).

Next, 4, the MPU 17 determines the forward scattered light intensity ■. , 90° scattered light intensity■,. A site duram is created in which the vertical and horizontal axes are respectively (Sr2, see FIG. 3(a)).

In Fig. 3(a), A is ghost and garbage of hemolyzed red blood cells;
L indicates lymphocytes, M indicates monocytes, and G indicates granulocytes.

Next, auto-trigger calculation is performed to group L lymphocytes.
A fraction containing only the above is set (Sr3).

There are various methods for this automatic trigger calculation, for example, Japanese Patent Application No. 63-193033 or Japanese Patent Application No. 63-19.
The method described in the specification of No. 3072 can be applied. Furthermore, in the case of abnormal data, it is also possible to manually set both divisions using a mouse or the like.

A histogram is created for the PE fluorescence intensity IP of the cell light information belonging to the fraction, that is, the cell light information of lymphocytes (Sr1, see FIG. 3(b)).

Since the monoclonal antibody 0KB7 selectively binds to lymphoid B cells, a group of B cells and a group of T cells appear separately as shown in FIG. 3(b). In Sr1, auto-trigger calculation is performed to set [p] and select the cell optical information of T cells.In Sr6, the perpendicular component polarization intensity r and the parallel component polarization intensity ■ of the T cell cell optical information obtained in this way are set. , each T
Calculate the degree of fluorescence polarization P for the cells. Then, a histogram is created for the polarization component intensities (1) and (4) I+ [see FIG. 4(a)], and a histogram of the fluorescence polarization degree P is created (see FIG. 4(b), 5T7). Furthermore, statistics such as the average value are calculated from the histogram of the degree of fluorescence polarization P (5T8), and these values are displayed on the CRT 19 (Sr9) and printed out on the printer 20 (STIO). Note that the average value of the histogram of the degree of fluorescence polarization P shown in FIG. 4(b) corresponds to the measurement result by the conventional cuhette method shown in FIG.

In the above explanation, fluorescence polarization analysis of lymphoid T cells was taken as an example, but the present invention is also applicable to fluorescence polarization analysis of lymphocytes B cells, monocytes, granulocytes, or other blood cells. It is possible.

(F) Bright effect As explained above, the cell fluorescence polarization analysis method of the first claim stains cells in a sample with at least a fluorescent dye related to polarization analysis, and the sample is placed in a thin liquid stream. The flowing cells are irradiated with a light beam to excite the fluorescence of the fluorescent dye, and for each cell, cell light information consisting of the intensity of the polarized component of the fluorescence and other parameters is detected. Using other parameters, the cell light information of the target cell population is sorted from the cell light information of other cell populations, and based on the intensity of the fluorescence polarization component of the cell light information of the selected target cell population, the target cell Because it analyzes the degree of fluorescence polarization of a population, it is easy to prepare samples, and the analysis accuracy is high.
Furthermore, it has the advantage of being able to finely select and analyze cell populations.

Further, the cell fluorescence polarization analyzer according to the second aspect of the present invention provides at least a flow cell in which a suspension of cells stained with a fluorescent dye related to polarization analysis is flowed, and a cell flowing through the flow cell that is provided with the fluorescent dye. a light source that irradiates a light beam that excites fluorescence; a cell light information detection means that detects cell light information consisting of the polarization component intensity of the fluorescence and other parameters for each cell irradiated with the light beam; Cell light information selection means for selecting cell light information of a target cell population from cell light information of other cell populations using the other parameters obtained by the cell light information detection means, and this cell light information selection means A cell fluorescence polarization degree analysis means for analyzing the degree of fluorescence polarization of the target cell population based on the polarization component intensity of the fluorescence of the cell light information of the target cell population selected by the method, and outputting the analysis results of this cell fluorescence polarization degree analysis means. Since the present invention is equipped with an output means for outputting information, it has the advantage that the cell fluorescence polarization analysis method according to the first aspect can be carried out easily.

[Brief explanation of drawings]

FIG. 1 is a flow diagram explaining the operation of a cell fluorescence polarization analyzer according to an embodiment of the present invention, FIG. 2 is a diagram explaining the configuration of the cell fluorescence polarization analyzer, and FIG. a) is
Forward scattered light intensity in the cell fluorescence polarization analyzer -
90° scattered light intensity cytogram side - A diagram showing an example, FIG. 3 (b) is a diagram showing an example of a histogram of PE fluorescence intensity in the same cell fluorescence polarization analyzer, and FIG. 4 (a) is
FIG. 4(b) is a diagram showing an example of a histogram of fluorescence polarization component intensity in the cell fluorescence polarization analyzer, and FIG. 5 is a diagram showing an example of a histogram of fluorescence polarization intensity in the cell fluorescence polarization analyzer. FIG. 6 is a diagram explaining the principle of fluorescence polarization analysis, and FIG. 6 is a diagram explaining a conventional fluorescence polarization analysis method. 1: Flow cell, 2: Laser light source, 12 Nigeran-Thomson prism, 13a, 13b13c, 13d-13e: Photodetector, 17: MPU, 19: CRT. 20: Printer. Patent Applicant OMRON Co., Ltd. Agent Patent Attorney Shin Nakamura Shigeru Nakamura Figure (a) h Figure (b) p h Figure (a) n Engineering 1 h Figure (b) h Figure

Claims (2)

[Claims] (1) Stain the cells in the sample with at least a fluorescent dye related to polarization analysis, flow the sample into a thin liquid stream, irradiate the flowing cells with a light beam to excite the fluorescence of the fluorescent dye, and For cells, cell light information consisting of the fluorescence polarization component intensity and other parameters is detected, and by using the other parameters of the cell light information, the cell light information of the target cell population is transferred to cells of other cell populations. A cell fluorescence polarization degree analysis method for sorting based on optical information and analyzing the degree of fluorescence polarization of a target cell population based on the polarization component intensity of fluorescence of the cell optical information of the selected target cell population. (2) at least a flow cell in which a suspension of cells stained with a fluorescent dye related to polarization analysis is flowed, and a light source that irradiates the cells flowing in the flow cell with a light beam that excites the fluorescence of the fluorescent dye; Cell light information detection means for detecting cell light information consisting of the polarized component intensity of the fluorescence and other parameters for each cell irradiated with the light beam; A cell light information sorting means for selecting cell light information of a target cell population from cell light information of other cell populations using parameters; A cell fluorescence polarization degree comprising a cell fluorescence polarization degree analysis means for analyzing the fluorescence polarization degree of a target cell population based on the polarization component intensity of the fluorescence, and an output means for outputting the analysis result of the cell fluorescence polarization degree analysis means. Analysis equipment.