CN116106275A

CN116106275A - Tumor site determination device, tumor site determination method, and storage medium

Info

Publication number: CN116106275A
Application number: CN202111328605.XA
Authority: CN
Inventors: 越石直孝; 大河内健吾
Original assignee: Ushio Suzhou Co ltd
Current assignee: Ushio Suzhou Co ltd
Priority date: 2021-11-10
Filing date: 2021-11-10
Publication date: 2023-05-12

Abstract

A tumor site determination device is provided with a light source unit (3), a spectroscopic unit (5), a light receiving unit (6), and a signal processing unit (7). The signal processing unit determines that the tumor site is included in the specimen if all of the following three conditions are satisfied: (1) the intensity of the first light having the first peak wavelength at the center wavelength, or the intensity of the fifth light and the sixth light having the center wavelength in the vicinity of the first peak wavelength is equal to or higher than a predetermined first peak threshold, (2) the spectrum showing the relationship between the intensity of the light emitted from the porphyrin after excitation and the wavelength has a shape having peaks in the vicinity of the first peak wavelength and the vicinity of the second peak wavelength and having valleys in the vicinity of the first valley wavelength and/or the vicinity of the second valley wavelength, (3) the larger one of the intensities of the fifth light and the sixth light is set as I _p1 The smaller one is designated as I _p2 Will I _p1 And I _p2 The percentage ratio of the intensity difference is set as R _Ip When R is _Ip Is above a certain value, wherein R _Ip ＝(1‑I _p2 /I _p1 )×100。

Description

Tumor site determination device, tumor site determination method, and storage medium

Technical Field

The present invention relates to a determination device, a determination method, and a storage medium for determining whether or not a tumor site is included in a sample taken from a patient by irradiating the sample with excitation light and detecting fluorescence emitted from porphyrins in the sample after excitation.

Background

In the prior art, there is a method of performing fluorescence observation using 5-aminolevulinic acid (5-ALA). 5-ALA is one of the amino acids also present in living bodies, is water-soluble, and can be orally and topically administered. When 5-ALA is administered from outside, it is rapidly metabolized to heme in normal cells, but relatively large amounts of porphyrins (e.g., protoporphyrin IX) as metabolites are selectively accumulated in cancer cells due to differences in the activities of metabolic enzymes. Here, since heme is not a fluorescent substance, and porphyrin is a fluorescent substance, it is possible to identify a tumor site and a non-tumor site by irradiating a substance to be observed with a predetermined excitation light and performing light-receiving analysis of fluorescence emitted from the substance. For example, in brain surgery, blue excitation light may be irradiated, and if cancer cells are present, porphyrins in the cancer cells may fluoresce in red under the blue excitation light, thereby visually grasping the extent of spread of the cancer cells.

In a human organism, in addition to porphyrins, some other healthy tissues (e.g., FAD (Flavin Adenine Dinucleotide, flavin adenine dinucleotide), fat, collagen (collagen), elastin (Elastin), NADH (nicotinamide adenine dinucleotide ), etc. also fluoresce under blue excitation light.

Therefore, for example, when a sample from a human body is irradiated with blue excitation light, it is necessary to distinguish whether fluorescence generated by excitation of the sample is derived from porphyrins or other healthy tissues having autofluorescence.

In contrast, in the prior art, there is known a method for determining porphyrins by utilizing spectral characteristics of porphyrins and other healthy tissues having autofluorescence, and as shown in fig. 1, porphyrins (protoporphyrin IX as an example thereof is shown in the figure) generally have a characteristic of having one peak in the vicinity of wavelengths 620nm to 635nm and 680 to 700nm, and FAD generally has a characteristic of decreasing fluorescence intensity smoothly with increasing wavelength in a region of 600nm or more. By utilizing the above-described characteristics of porphyrins, it is possible to determine whether or not a tumor site is included based on the shape characteristics of the spectrum by, for example, splitting light emitted from a patient sample and acquiring a spectrum representing a map of light intensity and wavelength.

For example, non-patent document 1 proposes a technique in which, after a spectrum of red fluorescence of protoporphyrin IX (hereinafter, also referred to as PpIX) as a porphyrin is observed, if a large peak near 620nm is confirmed, it is determined as positive.

Non-patent document 1:5-Aminolevulinic Acid-derived Tumor Fluorescence: the Diagnostic Accuracy of Visible Fluorescence Qualities as Corroborated by Spectrometry and Histology and Postoperative Imaging

Disclosure of Invention

However, the present inventors have studied many samples and have found that in the fluorescence spectrum from a sample to which 5-ALA is administered, a large peak is sometimes observed in the vicinity of 620nm to 635nm, but it is not necessarily positive (including cancer cells). For example, (a) and (b) of fig. 2 show fluorescence spectra of PpIX in two urine (samples) for examination of bladder cancer. Although large peaks were observed near 620nm, in practice, both of the samples were judged negative (excluding cancer cells) in pathological diagnosis.

Then, the present inventors have conducted a comparative study on the fluorescence spectra of porphyrins of a pathology-positive specimen and a pathology-negative specimen, and as a result, have found that, in the case where a peak shape generally occurring in the vicinity of 620nm to 635nm is deformed, the probability of being judged as negative in pathology diagnosis is high.

More specifically, the present inventors have found that, taking the above-described non-patent document 1 as an example, even when there is a large peak around 620nm in the fluorescence spectrum of protoporphyrin IX, if the difference in fluorescence intensity between 616nm and 635nm, which are predetermined two wavelengths around 620nm, is less than 22% (the peak height on the lower side is greater than 78% of the peak height on the upper side), the probability of being negative is high.

Further, the present inventors have found that, even when there is a large peak around 620nm in the fluorescence spectrum of protoporphyrin IX, although the difference in fluorescence intensity between 616nm and 635nm, which are predetermined two wavelengths around 620nm, is less than 22%, if the fluorescence intensity at a certain wavelength between 616nm and 635nm, which are predetermined two wavelengths around 620nm, is greater than these two wavelengths, the probability of positivity is high in some cases, taking the above-described non-patent document 1 as an example.

In view of the above findings, the present inventors have found that a determination device, a determination method, and a storage medium for determining whether or not a tumor site including a tumor site in a sample using the spectral characteristics of porphyrins can improve the matching rate (diagnosis accuracy) with pathological diagnosis.

The present invention provides a tumor site determination device, comprising: a light source unit that irradiates a sample with excitation light to excite porphyrins in the sample; a spectroscopic unit configured to separate light emitted from the porphyrin after excitation into at least second to sixth lights, the first light having a center wavelength of a first peak wavelength near a first peak of light intensity among fluorescence emitted from the porphyrin, the second light having a center wavelength of a first valley wavelength near a first valley of light intensity on a short wavelength side than the first peak of light intensity among fluorescence emitted from the porphyrin, the third light having a center wavelength of a second peak wavelength near a second peak of light intensity on a long wavelength side than the first peak of light intensity among fluorescence emitted from the porphyrin, the fourth light having a center wavelength of a second peak of light intensity near a second peak of light intensity on a long wavelength side than the first peak of light intensity, the fifth light having a center wavelength near the second valley of light intensity near the second peak of light intensity among fluorescence emitted from the porphyrin; light receiving parts for receiving the light passing through the light splitting parts in the light emitted by the porphyrin after excitation and converting the light into electric signals; and a signal processing unit configured to acquire, based on the electric signal obtained from the light receiving unit, at least second to sixth light splitting intensities among a first light splitting intensity indicating an intensity of the first light, a second light splitting intensity indicating an intensity of the second light, a third light splitting intensity indicating an intensity of the third light, a fourth light splitting intensity indicating an intensity of the fourth light, a fifth light splitting intensity indicating an intensity of the fifth light, and a sixth light splitting intensity indicating an intensity of the sixth light, the signal processing unit determining that the specimen includes a tumor site if all of the following three conditions are satisfied:

(1) The first or fifth or sixth spectral intensity is equal to or higher than a predetermined first peak threshold,

(2) A spectrum showing a relationship between the intensity and the wavelength of light emitted from the porphyrin after excitation has a shape having peaks near the first peak wavelength and near the second peak wavelength and having valleys near the first valley wavelength and/or near the second valley wavelength,

(3) The greater one of the fifth and sixth spectral intensities is defined as I _p1 The smaller one is designated as I _p2 Will I _p1 And I _p2 The percentage ratio of the intensity difference is set as R _Ip When R is _Ip Is above a certain value, wherein R _Ip ＝(1-I _p2 /I _p1 )×100。

In the present specification, "porphyrins" refer to compounds having a substituent on the porphyrin ring, and as one of porphyrins, there are, for example, other protoporphyrins such as photo-protoporphyrin (PPp) produced from PpIX, in addition to PpIX.

According to the present invention, sensitivity, specificity, and consistency with pathological diagnosis, that is, diagnosis accuracy (accuracy) can be improved as compared with the diagnosis result under pathological conditions.

Drawings

FIG. 1 is a diagram showing autofluorescence spectra of PpIX and FAD.

Fig. 2 (a) and (b) show fluorescence spectra of PpIX in two samples determined to be negative in pathological diagnosis, respectively.

Fig. 3 is a block diagram schematically showing an example of the internal configuration of the tumor site determination device of the present invention.

Fig. 4 is a flowchart showing the determination of a tumor site according to example 1.

FIG. 5 is a schematic view showing fluorescence spectra determined to be positive by example 2.

FIG. 6 is a schematic diagram showing the removal of Raman scattering from the spectrum of porphyrins by using curve fitting.

Fig. 7 is a schematic diagram showing another example of removing raman scattering from a spectrum of porphyrins by using curve fitting.

Fig. 8 is a schematic diagram showing another example of removing raman scattering from a spectrum of porphyrins by using curve fitting.

Fig. 9 is a schematic diagram showing another example of removing raman scattering from a spectrum of porphyrins by using curve fitting.

Description of the reference numerals

1, a sample; 2, an objective lens; 3 a light source part; a dichroic mirror 4; a light splitting unit; a light receiving unit; 7 an information processing unit; 8 display part.

Detailed Description

The constitution of the tumor site determination device of the present invention will be described with reference to the drawings. Fig. 3 is a block diagram schematically showing an example of the internal configuration of the tumor site determination device of the present invention.

The tumor site determination device of the present invention analyzes the spectrum of fluorescence emitted from porphyrins in sample 1 after excitation to determine whether or not the sample 1 contains a tumor site (cancer cells). The sample 1 is a biopsy material (for example, a biological tissue including sentinel lymph nodes) as a determination target for a tumor site. When the biopsy material contains a tumor site, porphyrins accumulate in the tumor site. As an example, the biopsy material may be a biopsy material that is removed after 5-ALA is administered to a human body, or may be a sample to which 5-ALA is added after removal. The method of taking out the biopsy material from the human body part suspected of cancer differs depending on the part suspected of cancer, and is not particularly limited, for example, taking out the biopsy material from the target part by a syringe in the case of breast cancer or thyroid cancer, taking out the biopsy material by a cervical brush in the case of cervical cancer, taking out the biopsy material from urine in the case of bladder cancer, taking out the biopsy material from saliva in the case of oral cancer, and taking out the biopsy material from sputum in the case of lung cancer. The sample can be obtained by placing a sample of the subject into a container and then performing a centrifugal motion so that a portion having higher transparency falls below the container. The sample 1 may be held by a sample holder, not shown, or may be loaded into a loading unit, not shown, or a container in which the sample is placed may be loaded into the loading unit.

As shown in fig. 2, the tumor site determination device includes an objective lens 2, a light source unit 3, a dichroic mirror (dichroic mirror) 4, a spectroscopic unit 5, a light receiving unit 6, an information processing unit 7, and a display unit 8.

The light source unit 3 may be constituted by, for example, a mercury lamp, a xenon lamp, a halogen lamp, a metal halide lamp, or the like, a light emitting diode element, a laser diode element, or the like. It may have an unillustrated filter having a function of selectively transmitting light of a specific wavelength among the light emitted from the light source section 3, and may be constituted by a dielectric multilayer film or the like, for example. The filter may have a function of selectively transmitting light in a wavelength band required for exciting porphyrins accumulated when a tumor site is included in a sample. As an example, the filter may have a function of selectively transmitting light in a specific wavelength band of 385nm to 425nm, or 500nm to 580 nm.

The dichroic mirror 4 has a function of reflecting light in a predetermined wavelength band and transmitting light in another predetermined wavelength band, and may be formed of, for example, a dielectric multilayer film. Here, the description will be given of the case where the dichroic mirror 4 has a function of reflecting light having a wavelength of 405nm and transmitting light having a wavelength of 580nm or more. In the case of having a filter, the dichroic mirror 4 may have a function of reflecting light having a wavelength selected by the filter and transmitting light having a wavelength which is emitted from the sample 1 and selected by each filter included in the spectroscopic unit 5 described later.

Excitation light 31 emitted from the light source unit 3 (or having a wavelength of 405nm transmitted through the filter) is reflected by the dichroic mirror 4 and introduced into the objective lens 2. Then, the light passed through the objective lens 2 is irradiated onto the sample 1. When a substance (for example, ppIX) as a porphyrin is accumulated in the sample 1, the substance is excited by the excitation light 31, and emits fluorescence 32.

The fluorescent light 32 travels in a direction opposite to the excitation light and is introduced into the objective lens 2. Then, the fluorescence 32 is transmitted from the dichroic mirror 4 and enters the spectroscopic unit 5.

In the present embodiment, the spectroscopic unit 5 may have a function of dispersing the fluorescence incident on the light receiving unit 6, and is preferably a spectroscopic unit that disperses the input light by a prism, a diffraction grating, or the like, and measures the dispersed light by a light receiving member to obtain a spectrum, or may be a plurality of filters that are provided on the optical path between the sample 1 and the light receiving unit 6 so as to be switchable by a filter switching unit. When a plurality of filters are used, each filter has a function of selectively transmitting light of a specific wavelength, and may be constituted by an optical filter such as a bandpass filter. In the present embodiment, a case where the spectroscopic unit 5 is a spectroscope will be described.

The spectroscopic unit 5 separates light emitted from the porphyrin after excitation into at least second to sixth lights, the first light having a center wavelength of a first peak wavelength near the first peak of the fluorescence emitted from the porphyrin, the second light having a center wavelength of a first trough wavelength near the first trough of the fluorescence emitted from the porphyrin at a shorter wavelength than the first peak, the third light having a center wavelength of a second peak wavelength near the second peak of the fluorescence emitted from the porphyrin at a longer wavelength than the first peak, the fourth light having a center wavelength of a second trough wavelength near the second peak of the fluorescence emitted from the porphyrin at a longer wavelength than the first peak, and the light intensity near the second trough of the fluorescence emitted from the porphyrin at a shorter wavelength than the second peak, and the fifth light and the sixth light each having a center wavelength near the first peak. Here, as the porphyrin, it is known that the fluorescence spectrum has different peaks in the cell, in an aqueous solution, or in an organic solvent, and the present invention exemplifies a fluorescence spectrum having steep first and second peaks in the vicinity of wavelengths 620nm and 680nm and PpIX having first and second peaks in the vicinity of wavelengths 600 and 650nm, that is, the first peak wavelength is 620nm, the second peak wavelength is 680nm, the first valley wavelength is 600nm, and the second valley wavelength is 650nm. In this case, the center wavelengths of the fifth light and the sixth light, which are predetermined two wavelengths around the first peak wavelength, are set to 616nm and 635nm, respectively. The reason why the predetermined two wavelengths are selected to be 616nm and 635nm is that the inventors have found that, in the case where a large peak exists near 620nm in the fluorescence spectrum of protoporphyrin IX but the difference in fluorescence intensity between 616nm and 635nm is less than 22% in the case where it is judged to be negative in the actual pathological diagnosis, the inventors have found that, in the case where the difference in fluorescence intensity between 616nm and 635nm is less than 22% in the pathological negative, there are also many cases where a small peak appears at 616nm and 635nm. In addition, the present inventors have found that the smaller the difference in fluorescence intensity at 616nm and 635nm is smaller than 22%, the smaller the peak is easily observed at 616nm and 635nm, respectively, and the larger the difference in fluorescence intensity at 616nm and 635nm is larger than 22%, the smaller the peak is easily observed between 616nm and 635nm. In addition, in consideration of the difference in reagent solution and device, the wavelength values around 616nm and 635nm, for example, 615nm, and the like may be selected. It is also understood that when the first peak is steep at the wavelength 620nm±3nm, the center wavelengths of the fifth light and the sixth light, which are predetermined two wavelengths around the first peak wavelength, are 616nm±3nm and 635nm±3nm, respectively.

The spectroscopic unit 5 may further separate the light emitted from the porphyrin after excitation into a seventh light, and the center wavelength of the seventh light may be arbitrarily selected from the center wavelength of the fifth light and the center wavelength of the sixth light, and may be the same as the center wavelength of the first light when the center wavelength is randomly selected.

The reason why the seventh light is split out is because the present inventors have found that, uniquely, irrespective of whether or not the difference in fluorescence intensity at 616nm and 635nm is smaller than 22%, if the fluorescence intensity at a certain wavelength between 616nm and 635nm is larger than those two wavelengths, the possibility of being judged to be positive in actual pathological diagnosis is high (for example, fig. 5).

The light receiving unit 6 may be any one or more of a photodiode, a photomultiplier, a photoresistor, a phototube, and a camera. In the case of using two or more of them, an array may be formed by arranging them in one or two dimensions, for example, and a high space resolution can be obtained. The light receiving unit 6 receives the fluorescent light 32 transmitted from the spectroscopic unit 5, and converts the received fluorescent light into an electric signal. The light receiving unit 6 outputs an electric signal to the information processing unit 7, and here, position information indicating a specific position of the fluorescent porphyrins in the sample 1 may be output to the information processing unit 7.

The information processing unit 7 acquires each of the spectroscopic intensities indicating the intensity of each of the fluorescent lights 32 based on the electric signal obtained from the light receiving unit 6, and determines whether or not the tumor site is included in the sample 1 based on the acquired spectroscopic intensity. The information processing unit 7 is constituted by, for example, a microcomputer, a CPU, or the like.

Specifically, the information processing unit 7 may process the electric signal obtained from the light receiving unit 6 to convert the electric signal into light intensity, and then determine whether or not the tumor site is contained in the sample 1 by a determination formula concerning the light intensity, which will be described later. The information processing unit 7 outputs the result of the determination to the display unit 8, and preferably connects the light intensities to map the spectrum showing the mapping of the light intensities and the wavelengths, so that the user can determine the degree of coincidence between the spectrum and the spectrum of porphyrins by visual confirmation.

The display unit 8 corresponds to a monitor that displays a determination result (for example, a result obtained by finally determining a tumor site and a non-tumor site) by the information processing unit, a spectrum indicating a map of light intensity and wavelength, and the like. Here, the configuration in which the display unit 8 is provided in the main body of the tumor site determination device is shown, but a configuration in which a display unit such as a monitor is provided in addition to the tumor site determination device without providing a display unit in the main body of the device may be adopted. When the positional information of the sample 1 is acquired, the display unit 8 may display image data obtained by, for example, marking or developing a tumor site at a predetermined position on the image of the sample based on the coordinate information of the tumor site sent from the information processing unit 7. If there is no region determined to be a tumor site by the information processing unit 7, the information of the content may be displayed on the display unit 8. By visually checking the display unit 8, the inspector can know whether or not a tumor site exists in the sample 1, and can easily know the existence site of the tumor site even when the positional information is acquired.

Next, a specific example of determining whether or not a tumor site is included in a sample by using the present apparatus will be described.

First, excitation light 31 of blue color having a wavelength of 405nm is emitted from a light source unit 3, the excitation light 31 is reflected by a dichroic mirror 4, is introduced into an objective lens 2, and is irradiated onto a sample 1, ppIX is accumulated as porphyrin in the sample 1, and is excited by the excitation light 31 to emit fluorescence 32, and the fluorescence 32 travels in a direction opposite to the excitation light, and is introduced into the objective lens 2. Then, the fluorescence 32 is transmitted from the dichroic mirror 4 and enters the spectroscopic unit 5.

The spectroscopic unit 5 splits the fluorescence 32 into first to seventh light beams, for example. The light receiving unit 6 receives the first to seventh lights transmitted from the spectroscopic unit 5, converts the lights into an electric signal, and sends the electric signal to the information processing unit 7. The information processing unit 7 obtains first to seventh spectral intensities indicating intensities of the first to seventh lights based on the electric signal obtained from the light receiving unit 6.

Then, the information processing unit 7 determines whether or not the tumor site is included in the sample using the first to seventh spectral intensities.

The following two examples are examples of specific modes for determining.

[ example 1 ]

The present inventors have found that it is possible to determine that a tumor site is included in a specimen as long as three conditions are satisfied:

(2) The spectrum showing the relationship between the intensity and the wavelength of light emitted from the porphyrin after excitation has a shape having peaks near the first peak wavelength and near the second peak wavelength and having valleys near the first valley wavelength and/or near the second valley wavelength,

(3) The larger one of the fifth and sixth spectral intensities is defined as I _p1 The smaller one is designated as I _p2 Will I _p1 And I _p2 The percentage ratio of the intensity difference is set as R _Ip When R is _Ip Is above a certain value, wherein R _Ip ＝(1-I _p2 /I _p1 )×100。

Next, the principle of using these three conditions will be specifically described.

As shown in fig. 1, for example, a porphyrin has a spectral feature having two peaks, and the fluorescence spectrum of PpIX, which is a porphyrin, has steep first and second peaks around wavelengths 620nm and 680nm, and usually has a first peak (fluorescence intensity) larger and a first and second valley at wavelengths 600 and 650nm, respectively, and the intensities of the first and second peaks are significantly lower than those of the first and second peaks. However, there are cases where only one trough is shown depending on the sample and the solution.

Here, the condition (1) is to confirm whether or not the fluorescence spectrum of the light emitted from the sample has a large first peak like porphyrins, and since the higher the concentration of porphyrins is, the higher the probability of containing cancer cells is, the more characteristic spectrum characteristic of porphyrins is exhibited, and thus, in fig. 4, for example, the spectrum of the sample having a fluorescence intensity smaller than the threshold value and thus representing less accumulated porphyrins is first excluded. Here, since the center wavelengths of the fifth light and the sixth light are set in the vicinity of the first peak wavelength, it is sufficient to determine at least one of the first spectral intensity, the fifth spectral intensity, and the sixth spectral intensity, and in the case where the first spectral intensity is not used, the first light may be omitted.

The condition (2) is to confirm whether or not the fluorescence spectrum of the light emitted from the sample has a shape having two peaks and one or two valleys like porphyrins. As an embodiment thereof, it can be confirmed whether the first spectroscopic intensity is greater than the third spectroscopic intensity, whether the third spectroscopic intensity is equal to or greater than a predetermined second peak threshold, whether the second spectroscopic intensity is equal to or less than a predetermined first valley threshold, whether the fourth spectroscopic intensity is equal to or less than a predetermined second valley threshold, whether the second spectroscopic intensity is less than the first spectroscopic intensity and/or the third spectroscopic intensity, whether the fourth spectroscopic intensity is less than the third spectroscopic intensity, or the like, as long as it can be confirmed whether the fluorescence spectrum of the light emitted from the sample has a shape having two peaks and one or two valleys like porphyrins, and therefore, in fig. 4, for example, the spectrum of FAD, which is a representative substance having autofluorescence in a human body, smoothly decreases as the wavelength becomes longer in a region of 600nm or more, is excluded because it does not have a shape having two peaks.

The above condition (3) is directed to the findings of the present inventors that the condition (1) (2) is satisfied, but the condition is judged negative in the actual pathological diagnosis, and the difference in fluorescence intensity at 616nm and 635nm is less than 22%. Then, in fig. 4, the spectrum having two smaller peaks at 616nm and 635nm around the first peak wavelength and having a difference in fluorescence intensity of less than 22% between the two will be excluded, so that in fig. 4, only the spectrum judged positive at the far right end remains.

Here, 22% is an example of the "constant value" in the condition (3), and can be appropriately adjusted according to the apparatus, the reagent solution, and the like, but is usually selected in the range of 15 to 30%. In practical applications, a so-called gray zone (gray zone) indicating "suspected positives" may be flexibly provided around 22%, for example, in the range of 20 to 24%.

[ example 2 ]

(4) The seventh spectroscopic intensity is greater than the fifth spectroscopic intensity and the sixth spectroscopic intensity.

Since the conditions (1) and (2) are the same as those in example 1, only the condition (4) will be described.

The condition (4) described above is directed to the finding of the present inventors that, irrespective of whether or not the difference in fluorescence intensity at 616nm and 635nm is smaller than 22%, if the fluorescence intensity at a certain wavelength between 616nm and 635nm is larger than those at both wavelengths, the possibility of being judged to be positive in actual pathological diagnosis is high. Thus, for example, as shown in FIG. 5, the fluorescence intensity (I) at 616nm (point A) _p1 ) And fluorescence intensity at 635nm (point B) (I _p2 ) The difference between (a) is small (much less than 22%), but the light intensity at point C between 616nm and 635nm (I _pmiddle ) Greater than I _p1 、I _p2 Will be judged as positive.

Here, it is more preferable that the spectrum shape between 616nm and 635nm has a smooth peak shape (parabolic shape) as a whole, that is, does not have fine wavy undulations, horizontal lines, or the like in a zigzag shape, as shown in fig. 5.

The

embodiments

1 and 2 described above can be carried out in various ways, and examples of the specific mode of determination include a so-called flow method.

In this flow method, there is an example in which a next condition is determined every time one condition is satisfied, a positive condition is determined until all conditions are satisfied, and a negative condition is determined by ending the program if any condition is not satisfied.

In the case of the flow method, the determination procedure of the above conditions is not limited as well.

In this flow method, it is also possible to first determine whether or not the sample is "not subjected to the operation for affecting the concentration of porphyrins", and to exclude the sample directly without performing the determinations of the conditions (1) to (4) when the determination is made that the operation for affecting the concentration of porphyrins is performed, which will be described in detail later.

(other embodiments)

Regarding porphyrins accumulated in tumor sites, ppIX (protoporphyrin IX) is exemplified above, but protoporphyrins which are the upper concepts of protoporphyrin IX and porphyrins which are the upper concepts of protoporphyrin IX are also characterized by having two peaks and two valleys, and the peaks and the valleys are respectively similar to the values of protoporphyrin IX, so that the principle of the present invention can be applied to determination of the concentration of any protoporphyrin or porphyrin other than protoporphyrin IX.

Specifically, since coproporphyrin III and uroporphyrin III have characteristics of having two peaks around a specific wavelength, respectively, and the peak and the trough are respectively similar to those of protoporphyrin IX, the concentration determination of coproporphyrin III and uroporphyrin III can be performed by the apparatus and method according to the present invention, and the center wavelength used for the spectroscopic by the spectroscopic unit 5 needs to be adjusted for different porphyrins.

The present invention is particularly applicable to a sample which has not been subjected to an operation that affects the concentration of porphyrins. This is because such an operation may cause the sample of the patient to deviate from the concentration of porphyrins to be measured in a normal state. Here, the operation affecting the concentration of porphyrins means, for example, any of hormone therapy, steroid (steroid) therapy, radiation therapy, cervical conization, administration of anticancer drugs, administration of iron agents, and administration of carbazochrome sodium sulfonate hydrate (Carbazochrome Sodium Sulfonate Hydrate).

Therefore, it is preferable that the patient to be examined is checked in advance for cancer surgery, administration history, and treatment history before the determination of the present invention, and the patient is not subjected to the operation that affects the concentration of porphyrins. For example, in the case where cervical cancer is to be diagnosed, a cone resection (Conization), radiation therapy (radiation therapy), or hormone replacement therapy (Hormone Replacement Therapy) may be applied to a patient, and in this case, the patient may not be the subject to be measured according to the present invention.

Instead of performing the above-described confirmation in advance, the signal processing unit may perform the judgment of the porphyrin based on one or more of the operation history, the administration history, the physiological cycle, and the age of the patient who is the sample after the judgment of whether the porphyrin is at a high concentration or a low concentration, so that it is possible to perform the more accurate cancer judgment, that is, to more accurately judge whether or not cancer cells are contained. For example, in the case of further determining based on the physiological cycle of the patient, it is possible to cite that the "ovulatory period" and the "luteal period" of the patient are each determined as one parameter.

The above-mentioned fluorescence spectra of PpIX have steep first and second peaks near wavelengths 620nm and 680nm and first and second valleys near wavelengths 600 and 650nm, but these wavelengths are merely examples, and are specific values of spectra commonly recognized and frequently detected from cancer cells in practical use. More strictly, ppIX may have different peaks in fluorescence spectra in cells, aqueous solutions, or organic solvents, and for example, depending on the reagent solutions, ppIX may have steep first and second peaks around 635nm and 700nm and first and second valleys around 600 and 655 nm. In addition, there are cases where the fluorescence spectrum of PpIX has a steep first peak around the wavelength 615nm due to the device, solution, or the like.

In addition, the above-described case where the spectroscopic unit is a spectroscope is exemplified, but a plurality of filters may be used as the spectroscopic unit. In this case, as an example of the conditions (1) to (3) that can be minimally determined, five filters having transmission center wavelengths of around 600, 616, 635, 650, and 680nm may be provided, and light emitted from the porphyrins after excitation may be transmitted through the five filters to be second light, fifth light, sixth light, fourth light, and third light, respectively.

Further, although each filter may be configured to transmit the first to seventh lights having a specific wavelength, it is preferable that the full width at half maximum of each filter is set to be as small as possible, for example, 10nm or less, without overlapping the wavelength ranges of lights that can be transmitted by each filter.

Further, it is more preferable that the information processing unit further removes Raman scattering (Raman scattering) generated when the light is irradiated to the sample after drawing a spectrum indicating a map of light intensity and wavelength.

Raman scattering is very weak and is not usually observed, but is a major source of spectral noise when porphyrins exhibit weak fluorescence depending on the sample. In addition, raman scattering cannot be removed by washing or reagents and attenuated.

As for raman scattering, which has a spectrum inherent to a substance, it generally has a shape having a peak around 470nm and a gentle bottom end of a slope as shown in fig. 6 (a), and the main raman scattering released from a sample is derived from an aqueous solution and takes on substantially the same shape even when the sample is different.

In this regard, the inventors uniquely thought to remove raman scattering from water when porphyrins are measured. Here, by using curve fitting (curve fitting), the raw data, which is the spectrum of porphyrins superimposed with raman scattering shown in fig. 6 (a), is processed to remove raman scattering.

As an example, first, as shown in fig. 6 b, a logarithmic curve (formula below) is generated so as to extend along the bottom of the downward slope of raman scattering, where a, b, and c are coefficients, λn is a wavelength, and the coefficients are not fixed but may be changed appropriately according to turbidity of the sample, composition of the solution, and the like.

Then, the above raw data is subjected to subtraction processing of subtracting the logarithmic curve, and thus, as shown in fig. 6 (c) and (d), the raman scattering portion is eliminated. Then, the spectrum of the remaining porphyrins may be further amplified so that the fluorescence amount of the porphyrins can be accurately obtained.

As an example, the linear interpolation-based method shown in fig. 7 may also be used. Specifically, as shown in fig. 7, a straight line is drawn so as to connect the ends of the desired signal, and then subtraction processing is performed to subtract the straight line from the above-described raw data, thereby eliminating the raman scattering portion.

As an example, the gaussian distribution-based method shown in fig. 8 may also be used. Specifically, as shown in fig. 8, the bottom of the downward slope of the raman scattering is made to coincide with the bottom of the downward slope of the gaussian distribution, and then subtraction processing is performed in the same manner as described above, thereby eliminating the portion of the raman scattering.

In addition, another method based on gaussian distribution as shown in fig. 9 may be used. Specifically, the raw data of the spectrum of the porphyrin superimposed with raman scattering shown in fig. 9 (a) is simulated using a plurality of gaussian distributions shown in fig. 9 (b), a synthetic curve for the raman scattering portion shown in fig. 9 (c) is synthesized, and then subtraction processing is performed in the same manner as described above, thereby eliminating the raman scattering portion.

Although the embodiments of the present invention have been described above, the embodiments are presented as examples and are not intended to limit the scope of the invention. These novel embodiments can be implemented in various other modes, and various omissions, substitutions, and changes can be made without departing from the spirit of the invention. These embodiments and modifications thereof are included in the scope and gist of the invention, and are included in the scope of the invention described in the claims and equivalents thereof.

Claims

1. A tumor site determination device is characterized by comprising:

a light source unit that irradiates a sample with excitation light to excite porphyrins in the sample;

a spectroscopic unit configured to separate light emitted from the porphyrin after excitation into at least second to sixth lights, the first light having a center wavelength of a first peak wavelength near a first peak of light intensity among fluorescence emitted from the porphyrin, the second light having a center wavelength of a first valley wavelength near a first valley of light intensity on a short wavelength side than the first peak of light intensity among fluorescence emitted from the porphyrin, the third light having a center wavelength of a second peak wavelength near a second peak of light intensity on a long wavelength side than the first peak of light intensity among fluorescence emitted from the porphyrin, the fourth light having a center wavelength of a second peak of light intensity near a second peak of light intensity on a long wavelength side than the first peak of light intensity, the fifth light having a center wavelength near the second valley of light intensity near the second peak of light intensity among fluorescence emitted from the porphyrin;

Light receiving parts for receiving the light passing through the light splitting parts in the light emitted by the porphyrin after excitation and converting the light into electric signals; and

a signal processing unit that acquires at least second to sixth light splitting intensities among a first light splitting intensity indicating an intensity of the first light, a second light splitting intensity indicating an intensity of the second light, a third light splitting intensity indicating an intensity of the third light, a fourth light splitting intensity indicating an intensity of the fourth light, a fifth light splitting intensity indicating an intensity of the fifth light, and a sixth light splitting intensity indicating an intensity of the sixth light, based on the electric signals obtained from the light receiving unit, wherein the signal processing unit determines that the specimen includes a tumor site if all of the following three conditions are satisfied:

2. A tumor site determination device is characterized by comprising:

a signal processing unit that acquires at least second to sixth light splitting intensities among a first light splitting intensity indicating an intensity of the first light, a second light splitting intensity indicating an intensity of the second light, a third light splitting intensity indicating an intensity of the third light, a fourth light splitting intensity indicating an intensity of the fourth light, a fifth light splitting intensity indicating an intensity of the fifth light, and a sixth light splitting intensity indicating an intensity of the sixth light, based on the electric signals obtained from the light receiving unit, wherein the signal processing unit determines that a tumor site is suspected to be included in the specimen if all of three conditions are satisfied:

(3) The greater one of the fifth and sixth spectral intensities is defined as I _p1 The smaller one is designated as I _p2 Will I _p1 And I _p2 The percentage ratio of the intensity difference is set as R _Ip When R is _Ip Within a range containing a certain value, wherein R _Ip ＝(1-I _p2 /I _p1 )×100。

3. A tumor site determination device is characterized by comprising:

a spectroscopic unit configured to separate light emitted from the porphyrin after excitation into at least second to seventh lights of first, second, third, fourth, fifth, sixth, and seventh lights, wherein a center wavelength of the first light is a first peak wavelength near a first peak in fluorescence emitted from the porphyrin, a center wavelength of the second light is a first valley wavelength near a first valley in fluorescence emitted from the porphyrin at a shorter wavelength side than the first peak, a center wavelength of the third light is a second peak wavelength near a second peak in fluorescence emitted from the porphyrin at a longer wavelength side than the first peak, a center wavelength of the fourth light is a second peak in fluorescence at a shorter wavelength side than the first peak, a light intensity near a second peak, and a center wavelength of the fifth light is a center wavelength between the fifth peak and the fifth peak, the center wavelength being a center wavelength between the fifth peak and the fifth peak;

a signal processing unit that acquires at least second to seventh light splitting intensities among a first light splitting intensity indicating an intensity of the first light, a second light splitting intensity indicating an intensity of the second light, a third light splitting intensity indicating an intensity of the third light, a fourth light splitting intensity indicating an intensity of the fourth light, a fifth light splitting intensity indicating an intensity of the fifth light, a sixth light splitting intensity indicating an intensity of the sixth light, and a seventh light splitting intensity indicating an intensity of the seventh light, based on the electric signal obtained from the light receiving unit, wherein the signal processing unit determines that the specimen includes a tumor site if all of the following three conditions are satisfied:

(4) The seventh spectral intensity is greater than the fifth and sixth spectral intensities.

4. The device for determining a tumor site according to any one of claim 1 to 3,

the light splitting part is provided with a plurality of optical filters.

5. The device for determining a tumor site according to claim 4,

the full width at half maximum of the plurality of filters is 10nm or less.

6. The device for determining a tumor site according to claim 4,

the optical path between the sample and the light receiving unit includes a filter switching unit for switching the plurality of filters so that any one filter is disposed in the optical path and the other filters exit the optical path.

7. The device for determining a tumor site according to any one of claim 1 to 3,

the porphyrins are protoporphyrins.

8. The device for determining a tumor site according to claim 7,

the protoporphyrin is protoporphyrin IX.

9. The device for determining a tumor site according to any one of claim 1 to 3,

the sample is not subjected to an operation that affects the concentration of porphyrins.

10. The device for determining a tumor site according to claim 9,

the operation affecting the concentration of porphyrins is any of hormone therapy, steroid therapy, radiation therapy, cervical conization, administration of anticancer drugs, administration of iron, and administration of carbazochrome sodium sulfonate hydrate.

11. The device for determining a tumor site according to any one of claim 1 to 3,

the light receiving part is one or more of a photodiode, a photomultiplier, a photoresistor, a phototube and a camera.

12. The device for determining a tumor site according to claim 11,

the light receiving portions are arranged in one or two dimensions using two or more of a photodiode, a photomultiplier tube, a photoresistor, a photocell, and a camera, thereby forming an array.

13. The device for determining a tumor site according to claim 4,

the signal processing unit processes the electric signal obtained from the light receiving unit and converts the electric signal into a spectrum indicating a map of light intensity and wavelength.

14. The device for determining a tumor site according to any one of claim 1 to 3,

The signal processing section removes raman scattering in the spectrum by curve fitting.

15. The device for determining a tumor site according to any one of claim 1 to 3,

the tumor site determination device further includes a loading unit for loading the specimen or a container in which the specimen is placed.

16. The device for determining a tumor site according to any one of claim 1 to 3,

the tumor part determination device further has a display for displaying the determination result of the signal processing unit.

17. The device for determining a tumor site according to any one of claim 1 to 3,

the beam splitter is a beam splitter.

18. A method for determining a tumor site, comprising the steps of:

an excitation step of exciting porphyrins in a sample by irradiating the sample with excitation light;

an obtaining step of obtaining at least second to sixth split intensities of a first split intensity indicating an intensity of a first light among lights emitted from the porphyrins after excitation, a second split intensity indicating an intensity of a second light among lights emitted from the porphyrins after excitation, a third split intensity indicating an intensity of a second light among lights emitted from the porphyrins after excitation, a fourth split intensity, a fifth split intensity, and a sixth split intensity, a center wavelength of the first light being a first valley wavelength on a shorter wavelength side than the first peak wavelength and an intensity of a third light among lights emitted from the porphyrins after excitation being a first valley wavelength, the center wavelength of the third light is a second peak wavelength near a second peak in the fluorescence emitted from the porphyrin, the light intensity being on a longer wavelength side than the first peak, the fourth light intensity representing the intensity of a fourth light in the light emitted from the porphyrin after excitation, the center wavelength of the fourth light being a second valley wavelength near a second valley in the fluorescence emitted from the porphyrin, the light intensity being on a longer wavelength side than the first peak and on a shorter wavelength side than the second peak, the fifth and sixth light intensities representing the intensities of a fifth and sixth light in the light emitted from the porphyrin after excitation, respectively, the center wavelengths of the fifth and sixth light being predetermined two wavelengths near the first peak; and

A determination step of determining that the tumor site is included in the specimen if all of the following three conditions are satisfied:

19. A method for determining a tumor site, comprising the steps of:

A determination step of determining that a tumor site is suspected to be contained in the specimen if all of the following three conditions are satisfied:

20. A method for determining a tumor site, comprising the steps of:

an obtaining step of obtaining at least second to seventh light-splitting intensities of a first light-splitting intensity representing an intensity of a first light of light emitted from the porphyrin after excitation, a second light-splitting intensity representing an intensity of a second light of light emitted from the porphyrin after excitation, a third light-splitting intensity representing an intensity of a second light of light emitted from the porphyrin after excitation, a fifth light-splitting intensity, a sixth light-splitting intensity, and a seventh light-splitting intensity, a center wavelength of the first light representing an intensity of a third light of light emitted from the porphyrin after excitation, the light intensity being near a first valley, a center wavelength of the second light being on a shorter wavelength side than the first peak, a center wavelength of the third light representing an intensity of the third light of light emitted from the porphyrin after excitation, the center wavelength of the third light is a second peak wavelength near a second peak in the fluorescence emitted from the porphyrin, the light intensity being on a longer wavelength side than the first peak, the fourth light intensity representing the intensity of a fourth light in the light emitted from the porphyrin after excitation, the center wavelength of the fourth light being a second valley wavelength near a second valley in the fluorescence emitted from the porphyrin, the light intensity being on a longer wavelength side than the first peak and on a shorter wavelength side than the second peak, the fifth and sixth light intensities representing the intensities of a fifth and sixth light in the light emitted from the porphyrin after excitation, respectively, the center wavelengths of the fifth and sixth light being predetermined two wavelengths near the first peak, the seventh spectral intensity represents an intensity of a seventh light of the light emitted from the porphyrin after excitation, the seventh light having a center wavelength between a center wavelength of the fifth light and a center wavelength of the sixth light; and

21. A computer-readable storage medium storing a program, the program when executed by a processor implementing the steps of:

22. A computer-readable storage medium storing a program, the program when executed by a processor implementing the steps of:

23. A computer-readable storage medium storing a program, the program when executed by a processor implementing the steps of: