JPH02168161A - Device for determining whether cells are benign or malignant - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed Description of the Invention] [Industrial Application Field] Can the present invention easily determine whether a cell is benign or malignant? Regarding i-placement.

[Prior art] Histopathological cancer diagnosis to date has basically been based on hematoxylin and eosin staining for paraffin tissue sections and Papanicolaou staining for cell #smears, and Diagnosis is based on optical microscopic examination, with the addition of specimens subjected to histochemical and immunohistochemical reactions, as appropriate.

On the other hand, the nuclear properties of cancer cells compared to those of normal cells.

(1) The nuclear morphology is irregular (=irregularly shaped), (2)
Nuclear volume is increased.

(3) Nuclear chromatin (composed of DNA and proteins) is highly stained and its distribution is irregular, (4) Nuclear DNA content is increasing. A regularization method has been proposed, and (a) in previous research using image analysis,
Normal staining (IE staining, Papanicolaou staining) specimens are subjected to morphological analysis and concentration analysis under m-weight of transmitted light using an optical microscope, and (b) DNA quantification on pathological specimens is performed using microfluorescence photometry waves. It's nine.

[Problems to be Solved by the Invention] However, this optical microscopic diagnosis is performed by pathologically determining "atypia as III tumor" regarding tissue and cell morphology, but it is subject to the subjective opinion of each pathologist. Because it relies heavily on physical and qualitative judgments, there has been a need for a more objective and quantitative method for determining whether a cell is benign or malignant. However, the conventional determination method using single-image analysis has not yet been put into practical use, and when attempting to search section specimens using microfluorophotometry, only the DNAa of nuclear fragments can be determined for the most part. Therefore, it is impossible to understand the proliferation activity by identifying DNA abnormalities specific to cancer cells, S-G, and stage cells.

The present invention has been made in view of these conventional problems, and an object of the present invention is to provide an objective device for determining whether a cell is benign or malignant even when a section sample is used. A further object of the present invention is to obtain a device for quantitatively evaluating the degree of irregularity in nuclear chromatin distribution in cancer cells.

[Means for Solving the Problems] In order to solve the above problems, the present invention uses a specimen in which the nuclear DNA of (+) cells is fluorescently stained with biochemical specificity to determine whether the cells are benign or malignant. In a device for determining.

A fluorescence microscope device that calculates the fluorescence intensity at the two-dimensional coordinate position of the fluorescence image of the DNA, and a fluorescence microscope device that develops the two-dimensional coordinate position of the nuclear DNA fluorescence image on a two-dimensional (X-Y) V plane and at each coordinate position. A device for determining whether a cell is benign or malignant, characterized in that it has a display device that displays a corresponding fluorescence intensity value as a pseudo three-dimensional solid figure extended along the vertical (Z) axis, and (2) ) A device for determining whether a cell is benign or malignant using a specimen in which minute nuclear DNA is fluorescently stained with biochemical specificity. a microscope device; and an instruction device that outputs No. 13 that identifies whether the nuclear DNA fluorescence image output as the video signal is of a normal cell or a cell of a search target site whose benign or malignant nature is to be determined. and inputting the video signal of the fluorescence microscope device and the identification signal of the instruction device, and identifying whether the nuclear DNA fluorescence image based on the manually generated video signal is of a normal cell or a cell of the search target site. , an image record J that stores a normal nuclear DNA fluorescence image and a fluorescence image of cells in the search target region, respectively.
Using the data stored in the a′\ stage and the lower image storage stage,
The parameters representing the irregularity of the nuclear chromatin distribution based on the nuclear DNA fluorescence image are set to the stored nuclear DNA.
a stage f for calculating the A fluorescence image; This is a cell benign/malignant determination device that determines whether cells in a search target region are benign or malignant.

[Action] In the present invention, the nucleus D, which plays a central role in the function of cells,
NA is fluorescently stained with biochemical specificity, and the benignity or malignancy of cells is objectively determined based on the irregularity of nuclear chromatin distribution based on the nuclear DNA fluorescence image obtained. Specifically, = (1) the two-dimensional coordinate position of each pixel constituting the nuclear DNA fluorescence image (position within the imaging plane of the camera)
is expanded onto a two-dimensional (X-Y) plane, and the fluorescence intensity values corresponding to each coordinate position are displayed as a pseudo-dimensional three-dimensional figure extending along the vertical (Z) axis, and the nuclear chromatin distribution represented by the surface structure is displayed. Based on the degree of pattern irregularity, it is possible to determine the degree of disturbance in the nuclear chromatin distribution of the cell, that is, whether the cell is benign or malignant. (2) Then, parameters are calculated to quantitatively evaluate the irregularity. , and whether those parameters regarding individual cells are conserved and distributed between the normal W8 fat group and the cell group at the search target site (there is no irregularity in the nuclear chromatin distribution of individual cells). It is possible to determine whether a cell is a cancer cell or not by quantitatively evaluating whether the cell is distributed in a divided manner (in this case, there is irregularity in the nuclear chromatin distribution of individual cells). Benign/malignant judgments can be made consistently and objectively.

[Example] The present invention will be described below based on the example shown in the drawings.

(b) Preparation of paraffin section specimens When preparing specimens, 4μ
Paraffin sections of m and 2 μl were serially
Cut 7N. The 4 μm section was used for optical microscopic observation.
The 2 μ field section is used for this analysis. The thickness of this 2μ peak is
This is the smallest size that can be sliced in order to capture a fluorescent image as clearly as possible and to prevent target cell nuclei from overlapping in the section as much as possible.

A normal HE-stained specimen is prepared from the 4#L-paraffin section. On the other hand, paraffin sections of ZuL
0PBS) solution at 37°C for 30 minutes to 1 hour], p
r staining [propidium 1odide 0.00
25 mg/ml (int, +ZX sodium citrate) staining solution at 4°C for 10 minutes] and encapsulation (0^PI.

Other DNA-specific fluorescent stains can also be applied), v
The former specimen is referred to as a PI-stained specimen, and the former HE-stained specimen is subjected to normal tissue observation using a separate optical microscope to determine a normal region (A) and a search target region (B). This operation is the same as that used in normal histopathological examination.

([1) Preparation of Frozen Section Specimens For frozen section specimens for rapid sectioning during surgery, the same technique as described above is carried out, except for the process of disassembly.

(8) Preparation of exfoliated cell smear (cell #) specimen Because exfoliated cell smear specimens cannot be obtained as continuous sections like paraffin section specimens, the specimen to be analyzed in one image is used individually. In other words, the exfoliated cell smear that is to be analyzed using a single method is smeared and fixed in the standard manner, then treated with RNase, and then treated with PI.
It is prepared by fluorescent staining.

(d) Imaging and processing of fluorescent images FIG. 1 is a block diagram of an embodiment of the present invention in which a PK-stained specimen is imaged and processed.

The microscope main body tLt is a well-known one, and allows selection of single-phase contrast observation and epifluorescence observation (G excitation in the case of PI staining).

The autostage 2 has a well-known configuration in which the specimen is left in place and is moved by the driving force of a motor within the X-Y plane perpendicular to the optical axis of the objective lens of the microscopic body l. To read coordinate values in the x-y plane (X-Y coordinate values).

The stage 2 has a built-in positive encoder, and the PAIN and P(x, y) signals are sent to the computer 5. Although this position encoder may be of an absolute type, it is convenient to use an incremental type with a home position signal to increase the resolution, and to detect the home position signal in response to a command from the computer 5 as an initial operation when the power is turned on. SIT tube type CCTV camera 3
takes an image of the specimen formed by the microscope main body 1 and sends it out as a video signal. The image processing unit 4 manually processes the video signal sent from the camera 3, classifies the image data of the normal region and the search target region in the image within one frame based on instructions from the computer 5, and stores it in the memory. do. The computer 5 integrally controls the autostage 2, the microscope main body 1, the camera 3, and the image processing unit 4 in accordance with the command from the command board 6, and causes the display device 7 to display the extinction results. Further, the computer 5 inputs the tS (coordinate signal) of the X-Y coordinate position from the autostage 2, and reads image data from the image processing unit 4.

With this configuration, the examiner must first check the microscope body l.
(instructs the command board 6 to enter the phase difference observation state, and as a result, the computer 5 switches the optical system of the microscope body 1 of the microscope 11 to form a phase difference optical system), and then,
Switch the auto stage 2 to manual mode (by instructing the command board 6, the computer 5 switches the auto stage 2 from automatic mode to manual mode) and operate the X and Y direction movement command members of stage 2 of the command boat 6. Then, the stage 2 is moved within the XY plane.

Then, a normal region (A) and a search target region (B) are determined under phase contrast observation. At this time, the normal region (A) and the search target region (B) are specified at multiple locations. It is only necessary to move (B) and issue commands to the command board 6 to take in data separately from (A) and (B).

As a result, the computer 5 sequentially numbers the data (A) and (B) input to the command board 6 (A
i; i=1~m, Bj; j=1~n),
The X-Y coordinates (jI) corresponding to each data Ai and Bj are stored in correspondence with each other according to the coordinate signal from the stage 2.

Next, the examiner issues a command to the command boat 6 to switch the microscope main body 1 from the above-mentioned phase difference observation to epifluorescence observation. As a result, according to the command from the computer 5, the microscope main body 1 switches the optical system to form an optical system for epi-fluorescence.

Hereinafter, the computer 5 shown in No. 212 (A) and (B)
The operation will be explained based on the flowchart. First, the examiner must press the measurement start switch on the command board 6. Then, the computer 5 first sequentially reads out the coordinate values corresponding to each of the normal parts (A) stored in the memory, that is, first, the coordinate values (A i In order to read (x, y), set i = 1 (Step 20), and read the standard value (A I ) CX, y) of the normal region AI stored in the memory.
) is read out (step 21).

Then, the stage is moved so that the read coordinates (N A l (x, y) and the coordinate values P (X, y) from the autostage 2 match (step 22), and when both coordinate values match (step 23), image processing unit 4
The image data of the nuclear DNA fluorescence image of the normal site A1 is stored in the memory (step 24). Since the image processing unit 4 has a frame memory, when the computer 5 detects that both coordinate values match in step 23, it instructs the image processing unit 4 to store the image data in the frame memory, Image data is thereby stored in the frame memory. The image processing unit 4 cuts out a nuclear DNA fluorescence image in the center from the image data in the frame memory, and stores the fluorescence intensity value in correspondence with the x-YW target value within the screen. In step 25, the computer 5
It is determined whether all the processes for the group of normal parts (A) stored in advance have been completed (the processes from step 21 to step 24 for each of AI to AII+), and if not, step 26 is performed for the current surprise. Add 1z7
Then, the process returns to step 21, and if one count is reached, j=x is set in step 27.

Then, the locus 1jg411 (B 1 (x, y)) of the search target site B1 of the group of search target sites (B) whose cancer is to be determined stored in the memory is read out (step 28). The read coordinate values (B
1 (x, y)) and the coordinate value P from autostage 2
Move the stage so that (x, y) becomes -・(
Step 29), when both coordinate values match (Step 30
), the nuclear DNA of the search target site B1 is sent to the image processing unit 4.
The image data of the fluorescent image is stored (step 31). The operation of the image processing unit 4 is the same as in the case of the normal region (A) described above.

Then, the computer 5 executes all the processes (from Bl to Bn
If the processing from step 28 to step 31) is completed or not, it is determined in step 32), and if it is not completed, 1 is added to the current j in step 33, and the process returns to step 28 to complete the process. For example, image processing unit 4
Arithmetic processing is performed using the stored data.

That is, the nucleus D of each site (AI to Am) of the group of normal sites CA), which is the memory data of the image processing unit 4.
NA fluorescence image data and 6g DN A'! of each site (Bl to Bn) in the group of search target site (B). :
From the data of one light image, the following parameters: (1) Nucleus D
Area of NA fluorescence image (2) Total fluorescence intensity at each contact, which is calculated as the sum of the fluorescence intensity of each pixel of the nuclear DNA fluorescence image to be analyzed, (3) (Fluorescence intensity of each pixel) / (-pixel (Mo average intensity per pixel) Y average DNA fluorescence intensity per pixel (4) 1 DNA fluorescence intensity of each pixel - P - sum of average intensity 1 per pixel (5) 1 (4) + 2 The total sum (6) (5), / (3) (7) (5) / (2) (8) CV (coefficient of variation of the fluorescence intensity value of each pixel) is calculated and used as calculation data (step 34).

Then, the data regarding the nuclear DNA fluorescence image of the cell group in the normal site (A) and the nuclear DNA fluorescence image of the cell group in the search target site (B) are compared, and the calculated data of the true person and the calculated data of the latter are compared. If any of the items (1) to (8) above are clearly divided into separate groups, for example, the calculated data of the former takes a value smaller than a certain value with respect to the item (4) above. When the calculation data from the past is scattered so that it takes a value larger than the value indicated in -F for the same item, the computer 5 determines whether it is a cell group or cancer cells in the search target region (B). If it is determined that there is a problem (malignant) and calculation data for all of the above items is preserved, the search target site (B
) is determined to be non-cancerous (benign), and that line is displayed on the display device 7 (steps 35 and 36).

In this way, when a search target site is suspected to have a neoplastic change and attempted to diagnose whether it is cancerous or not (benign or malignant), it is now possible to objectively determine and display whether or not it is cancerous through quantitative evaluation. Become.

The above items (1) to (8) were selected to fix the morphology of the nuclear chromatin distribution pattern based on the nuclear DNA fluorescence image, or were the items (1) to (8) calculated to determine the nuclear without quantitatively assessing the irregularity of the chromatin distribution pattern.

From the data stored in the image processing unit 4, the position information (x-y coordinate values on the frame memory) of each pixel is converted into two-dimensional (X
- Display the nuclear fluorescence image imaged in a plane as a three-dimensional figure in a pseudo three-dimensional display on the display device 2 by expanding it on a Y) plane and extending the fluorescence intensity value of each pixel along the vertical (Z) axis. In this case, by visually comparing the irregularity of the surface structure between the normal site and the search target site, it is possible to easily determine the degree of disorder in the lomatin distribution that is characteristic of cancer cells. can be evaluated.

That is, while the surface of the nuclear chromatin distribution pattern of normal cells is smooth, the surface of the nuclear chromatin distribution pattern of cancer cells is greatly disordered.

Therefore, if a cell at a search site for cancer diagnosis shows a nuclear chromatin distribution with a highly disordered surface, based on that irregularity, it is assumed that this is a cancer cell (i.e. , malignant). It has been confirmed that such determination results based on water droplets are almost consistent with histopathological diagnosis results for the more than 100 cases searched so far.

It should be noted that it is not essential that the display device 7 displays the patterns of the normal region and the search region together, and it is sufficient to display the cell groups of the search target region one after another in one minute to see the degree of irregularity of the surface structure. Since the display can be interrupted, the chromatin distribution pattern may be displayed in a pseudo three-dimensional manner only for the search site.

In addition, without having the computer 5 quantitatively judge the degree of irregularity of the chromatin distribution pattern, the above item (1)
Among the data from (8), eight types of specific two sets may be created, and a distribution graph of each obtained data may be displayed on the display device 217. In this case, the difference between the normal region (A) and the search target region (B) is further visually emphasized (recognized as a pattern rather than a mere numerical comparison), allowing for more accurate determination. .

An example of this case is shown in FIGS. 3 and 4.

Figures 3 and 4 show the analysis results for human gastric cancer, and the numbers (1) to (8) on the horizontal and vertical axes of each graph correspond to the parameter numbers (1) to (8) described in E, respectively. Corresponds (however, parameter (3) is for calculating parameter (6), so it does not appear on the first surface), 31A,
In FIG. 4, the black circles are data regarding individual cells from the normal site, and the plus (+) marks are data regarding individual cells from the search site.

In Figure 3, the distribution of black circles and the distribution of plus (+) marks form clearly different regions in each graph, and the cell groups in the search target region that have such a tendency are
All or most of the cells can be determined to be cancer cells. As mentioned above, this case was histopathologically diagnosed as gastric cancer.

Also, in Figure 4, the distribution of black circles and the distribution of plus (+) marks overlap in some graphs, but the general trend is that they form different regions, so this is the case. Most of the cell groups at the search target site that have a similar tendency are determined to be cancer cells. This case was also histopathologically diagnosed as adenocarcinoma of the stomach.

Furthermore, the results determined by displaying the analysis data as shown in Figures 3 and 4 mentioned above are based on histopathological diagnosis results made by several extremely reliable and experienced pathologists, and All of the more than 100 cases searched were consistent.

[Effect of the Invention 1 As described above, according to the present invention, whether or not a cell is a canal cell can be determined using quantitative parameters, pseudo three-dimensional display, or two-dimensional Since it can be determined based on the graph display,
It is possible to make cancer diagnosis objective.

In particular, when calculating parameters representing irregularity in nuclear chromatin distribution, the device can automatically determine the degree of irregularity in nuclear chromatin distribution characteristic of cancer cells. , it is possible to obtain a determination device that performs a completely objective determination without any room for the will of the examiner.

[Brief explanation of the drawing]

1 [A is a block diagram of one embodiment of the tree 511 light, FIGS. 2(A) and 2(B) are flow charts of the computer used in FIG. 1, and FIGS. 3 and 4 are specific parameters FIG. 6 is a diagram illustrating an example of a graph display for comparing the results of each cell group in the tE regular site and the search target site and determining whether or not the cell group is a canal cell. [Description of symbols of main parts] i ---11 fluorescence microscope body 2 --- CCTV camera, 4-111 image processing unit. 5-111 computer, 6--m-command boat, 7--1 display device no--funeral map

Claims (1)

[Scope of Claims] 1. In an apparatus for determining whether a cell is benign or malignant using a specimen in which the nuclear DNA of the cell is fluorescently stained with biochemical specificity, the two-dimensional coordinate position of the fluorescent image of the nuclear DNA; a fluorescence microscope device for determining the fluorescence intensity in the nuclear DNA fluorescence image; A device for determining whether a cell is benign or malignant, comprising: a display device that displays a stretched, pseudo-three-dimensional three-dimensional figure; 2. A fluorescence microscope that outputs a two-dimensional fluorescent image based on nuclear DNA as a video signal, in a device for determining whether a cell is benign or malignant using a specimen in which the nuclear DNA of the cell is fluorescently stained with biochemical specificity. an instruction device that outputs a signal that identifies whether the nuclear DNA fluorescence image output as the video signal is a normal cell or a cell to be searched to determine whether it is cancerous; and the fluorescence microscope. The video signal of the device and the identification signal of the instruction device are input, and it is determined whether the nuclear DNA fluorescence image based on the input video signal is of a normal cell or a cell in the search target region, and the nuclear DNA of the normal cell is identified. an image storage means for storing a plurality of fluorescence images and a plurality of nuclear DNA fluorescence images of cells at the search target site; a calculation means for calculating all stored nuclear DNA fluorescence images; A device for determining whether a cell group in a search target region is benign or malignant based on whether the cell group is benign or malignant.